Modulators of odorant receptors

ABSTRACT

The present invention relates to polypeptides capable of promoting odorant receptor cell surface localization and odorant receptor functional expression. The present invention further provides assays for the detection of ligands specific for various odorant receptors. Additionally, the present invention provides methods of screening for odorant receptor accessory protein polymorphisms and mutations associated with disease states, as well as methods of screening for therapeutic agents, ligands, and modulators of such proteins.

This application is a Divisional of U.S. patent application Ser. No.11/156,516, filed Jun. 20, 2005, which claims priority to U.S.Provisional Application Ser. No. 60/581,087, filed Jun. 18, 2004, andU.S. Provisional Application Ser. No. 60/582,011, filed Jun. 22, 2004,each of which is herein incorporated by reference in their entireties.

This invention was made with government support under Grants No. DC05782awarded by the National Institutes of Health.

FIELD OF THE INVENTION

The present invention relates to polypeptides capable of promotingodorant receptor cell surface localization and odorant receptorfunctional expression. The present invention further provides assays forthe detection of ligands specific for various odorant receptors.Additionally, the present invention provides methods of screening forodorant receptor accessory protein polymorphisms and mutationsassociated with disease states, as well as methods of screening fortherapeutic agents, ligands, and modulators of such proteins.

BACKGROUND OF THE INVENTION

Olfactory dysfunction arises from a variety of causes and profoundlyinfluences a patient's quality of life. Approximately 2 millionAmericans experience some type of olfactory dysfunction. Studies showthat olfactory dysfunction affects at least 1% of the population underthe age of 65 years, and well over 50% of the population older than 65years. The sense of smell determines the flavor of foods and beveragesand serves as an early warning system for the detection of environmentalhazards, such as spoiled food, leaking natural gas, smoke, or airbornepollutants. The losses or distortions of smell sensation can adverselyinfluence food preference, food intake and appetite.

Olfactory disorders are classified as follows: 1) anosmia: inability todetect qualitative olfactory sensations (e.g., absence of smellfunction), 2) partial anosmia: ability to perceive some, but not all,odorants, 3) hyposmia or microsmia: decreased sensitivity to odorants,4) hyperosmia: abnormally acute smell function, 5) dysosmia (cacosmia orparosmia): distorted or perverted smell perception or odorantstimulation, 6) phantosmia: dysosmic sensation perceived in the absenceof an odor stimulus (a.k.a. olfactory hallucination), and 7) olfactoryagnosia: inability to recognize an odor sensation.

Olfactory dysfunction is further classified as 1) conductive ortransport impairments from obstruction of nasal passages (e.g., chronicnasal inflammation, polyposis, etc.), 2) sensorineural impairments fromdamage to neuroepithelium (e.g., viral infection, airborne toxins,etc.), 3) central olfactory neural impairment from central nervoussystem damage (e.g., tumors, masses impacting on olfactory tract,neurodegenerative disorders, etc.). These categories are not mutuallyexclusive. For example, viruses can cause damage to the olfactoryneuroepithelium and they may also be transported into the centralnervous system via the olfactory nerve causing damage to the centralelements of the olfactory system.

Smelling abilities are initially determined by neurons in the olfactoryepithelium, the olfactory sensory neurons (hereinafter “olfactoryneurons). In olfactory neurons, odorant receptor (hereinafter “OR”)proteins, members of the G-protein coupled receptor (hereinafter “GPCR”)superfamily, are synthesized in the endoplasmic reticulum, transported,and eventually concentrated at the cell surface membrane of the cilia atthe tip of the dendrite. Considering that ORs have roles in targetrecognition of developing olfactory axons, OR proteins are also presentat axon terminals (see, e.g., Mombaerts, P., (1996) Cell 87, 675-686;Wang, F., et al. (1998) Cell 93, 47-60; each herein incorporated byreference in their entireties). In rodents, odorants are transduced byas many as 1000 different ORs encoded by a multigene family (see, e.g.,Axel, R. (1995) Sci Am 1273, 154-159; Buck, L., and Axel, R. (1991) Cell65, 175-187; Firestein, S. (2001) Nature 413, 211-218; Mombaerts, P.(1999) Annu Rev Neurosci 22, 487-509; Young, J. M., et al., (2002) HumMol Genet 11, 535-546; Zhang, X., and Firestein, S. (2002) Nat Neurosci5, 124-133; each herein incorporated by reference in their entirety).Each olfactory neuron expresses only one type of the OR, forming thecellular basis of odorant discrimination by olfactory neurons (see,e.g., Lewcock, J. W., and Reed, R. R. (2004) Proc Natl Acad Sci USA;Malnic, B., et al., (1999) Cell 96, 713-723; Serizawa, S., et al.,(2003) Science 302, 2088-2094; each herein incorporated by reference intheir entirety).

What is needed is a better understanding of olfactory sensation. What isfurther needed is a better understanding of odorant receptor function.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides capable of promotingodorant receptor cell surface localization and odorant receptorfunctional expression. The present invention further provides assays forthe detection of ligands specific for various odorant receptors.Additionally, the present invention provides methods of screening forodorant receptor accessory protein polymorphisms and mutationsassociated with disease states, as well as methods of screening fortherapeutic agents, ligands, and modulators of such proteins.

In preferred embodiments, the present invention provides a method foridentifying an odorant receptor ligand, comprising the steps of a)providing i) a cell line or cell membranes thereof comprising an odorantreceptor and a reporting agent, and ii) a test compound; b) exposing thetest compound to the cell line; and c) measuring the activity of thereporting agent. In some embodiments, the cell line expresses REEP1,RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1,RTP1-D1, RTP-D2, and RTP1-D3. In some embodiments, the cell line is aheterologous cell line or a natural cell line. In some embodiments, thecell line is a 293T cell line. In preferred embodiments, the odorantreceptor is a human odorant receptor. In other preferred embodiments,the test compound is an odiferous molecule. In even further embodiments,the reporting agent is regulated by a cAMP responsive element. Inpreferred embodiments, the cell line further comprises G_(αolf). Inother embodiments, the odorant receptor is a murine odorant receptor. Inother embodiments, the odorant receptor is a synthetic odorant receptor.In preferred embodiments, the odorant receptor comprises S6/79, S18,S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and/or MOR32-11. In otherembodiments, the reporting agent is an illuminating agent. In even otherembodiments, the illuminating agent is luciferase. In alternateembodiments, the method further comprises the step of detecting thepresence or absence of an odorant receptor ligand based upon thereporting agent activity.

In preferred embodiments, the present invention provides a cell lineexpressing an odorant receptor, wherein the expression is localized tothe cell surface. In preferred embodiments, the cell line comprises aheterologous gene. In preferred embodiments, the heterologous genecomprises one or more of REEP1, RTP1, and RTP2. In other preferredembodiments, the cell line is a 293T cell line. In some embodiments, theodorant receptor is a human odorant receptor. In preferred embodiments,the odorant receptor is tagged with a reporting agent. In someembodiments, the reporting agent is an illuminating reporting agent. Insome embodiments, the illuminating reporting agent comprisesglutathione-S-transferase (GST), c-myc, 6-histidine (6X-His), greenfluorescent protein (GFP), maltose binding protein (MBP), influenza Avirus haemagglutinin (HA), β-galactosidase, or GAL4. In preferredembodiments, the cell line further comprises G_(αolf) expression. Inpreferred embodiments, the odorant receptor is a murine odorantreceptor. In some embodiments, the odorant receptor is a syntheticodorant receptor. In preferred embodiments, the odorant receptorcomprises S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 andMOR32-11.

The present invention further provides an isolated nucleic acidcomprising a sequence encoding a protein comprising SEQ ID NOs: 21, 27,33, 34, 37, 38, and 41-50, and variants thereof that are at least 80%identical to SEQ ID NOs: 21, 27, 33, 34, 37, 38, and 41-50. In preferredembodiments, the sequence is operably linked to a heterologous promoter.In preferred embodiments, the sequence is contained within a vector. Inpreferred embodiments, the vector is within a host cell.

The present invention also provides isolated and purified nucleic acidsequences that hybridize under conditions of high stringency to anucleic acid comprising SEQ ID NOs: 1, 7, 13, 14, 17 and/or 18. Inpreferred embodiments, the sequence is operably linked to a heterologouspromoter. In preferred embodiments, the sequence is contained within avector. In some embodiments, the host vector is within a host cell. Infurther preferred embodiments, the host vector is expressed in a hostcell. In preferred embodiments, the host cell is located in an organism,wherein the organism is a non-human animal. In preferred embodiments,the present invention provides a polynucleotide sequence comprising atleast fifteen (e.g., 15, 18, 20, 21, 25, 50, 100, 1000, . . . )nucleotides capable of hybridizing under stringent conditions to theisolated nucleotide sequence.

In preferred embodiments, the present invention provides a polypeptideencoded by a nucleic acid selected from the group consisting of SEQ IDNOs: 1, 7, 13, 14, 17 and 18 and variants thereof that are at least 80%identical to SEQ ID NOs: 1, 7, 13, 14, 17 and 18. In furtherembodiments, the protein is at least 90% identical to SEQ ID NOs: 1, 7,13, 14, 17 and 18. In even further embodiments, the protein is at least95% identical to SEQ ID NOs: 1, 7, 13, 14, 17 and 18.

In preferred embodiments, the present invention provides a compositioncomprising a nucleic acid that inhibits the binding of at least aportion of a nucleic acid selected from the group consisting of SEQ IDNOs: 1, 7, 13, 14, 17 and 18 to their complementary sequences.

In preferred embodiments, the present invention provides a method fordetection of a variant REEP polypeptide in a subject, comprisingproviding a biological sample from a subject, wherein the biologicalsample comprises a REEP polypeptide; and detecting the presence orabsence of a variant REEP polypeptide in the biological sample. Inpreferred embodiments, the biological sample is selected from the groupconsisting of a blood sample, a tissue sample, a urine sample, and anamniotic fluid sample. In further embodiments, the subject is selectedfrom the group consisting of an embryo, a fetus, a newborn animal, and ayoung animal. In further embodiments, the animal is a human. Inpreferred embodiments, the detecting comprises differential antibodybinding. In further embodiments, the detection comprises a Western blot.In some preferred embodiments, the variant REEP polypeptide is a variantREEP1 polypeptide. In further embodiments, the detecting comprisesdetecting a REEP1 nucleic acid sequence.

In preferred embodiments, the present invention provides a method fordetection of a variant RTP polypeptide in a subject, comprisingproviding a biological sample from a subject, wherein the biologicalsample comprises a RTP polypeptide; and detecting the presence orabsence of a variant RTP polypeptide in the biological sample. Inpreferred embodiments, the biological sample is selected from the groupconsisting of a blood sample, a tissue sample, a urine sample, and anamniotic fluid sample. In further embodiments, the subject is selectedfrom the group consisting of an embryo, a fetus, a newborn animal, and ayoung animal. In further embodiments, the animal is a human. Inpreferred embodiments, the detecting comprises differential antibodybinding. In further embodiments, the detection comprises a Western blot.In some preferred embodiments, the variant RTP polypeptide is a variantRTP1 and/or RTP2 polypeptide. In further embodiments, the detectingcomprises detecting a RTP1 and/or RTP2 nucleic acid sequence. Inpreferred embodiments, the RTP1 variant is selected from the groupconsisting of RTP1-A1, RTP1-D1, and RTP1-D3.

In preferred embodiments, the present invention provides a kitcomprising a reagent for detecting the presence or absence of a variantREEP polypeptide in a biological sample. In some embodiments, the kitfurther comprises instruction for using the kit for the detecting thepresence or absence of a variant REEP polypeptide in a biologicalsample. In preferred embodiments, the REEP polypeptide is a REEP1polypeptide. In other embodiments, the REEP polypeptide is selected fromthe group consisting of REEP1-6. In preferred embodiments, theinstructions comprise instructions required by the U.S. Food and DrugAgency for in vitro diagnostic kits. In preferred embodiments, thereagent is one or more antibodies. In preferred embodiments, thebiological sample is selected from the group consisting of a bloodsample, a tissue sample, a urine sample, and an amniotic fluid sample.In preferred embodiments, the reagents are configured to detect a REEP1nucleic acid sequence.

In preferred embodiments, the present invention provides a kitcomprising a reagent for detecting the presence or absence of a variantRTP polypeptide in a biological sample. In some embodiments, the kitfurther comprises instruction for using the kit for the detecting thepresence or absence of a variant RTP polypeptide in a biological sample.In preferred embodiments, the RTP polypeptide is a RTP1 and/or RTP2polypeptide. In other embodiments, the RTP polypeptide is selected fromthe group consisting of RTP1-4. In preferred embodiments, theinstructions comprise instructions required by the U.S. Food and DrugAgency for in vitro diagnostic kits. In preferred embodiments, thereagent is one or more antibodies. In preferred embodiments, thebiological sample is selected from the group consisting of a bloodsample, a tissue sample, a urine sample, and an amniotic fluid sample.In preferred embodiments, the reagents are configured to detect a RTP1and/or RTP2 nucleic acid sequence. In preferred embodiments, the RTP1polypeptide is a variant RTP1 polypeptide selected from the groupconsisting of RTP1-A1, RTP1-D1, and RTP1-D3.

In preferred embodiments, the present invention provides a method forscreening compounds, comprising providing a sample expressing aheterologous REEP polypeptide and a test compound; and exposing thesample to the test compound and detecting a biological effect. Inpreferred embodiments, the REEP polypeptide is selected from the groupconsisting of REEP1-6. In preferred embodiments, the sample comprises acell. In preferred embodiments, the sample comprises a tissue. Inpreferred embodiments, the sample is found in a subject. In someembodiments, the biological effect comprises a change in activity ofREEP. In some embodiments, the biological effect comprises a change inexpression of REEP.

In preferred embodiments, the present invention provides a method forscreening compounds, comprising providing a sample expressing aheterologous RTP polypeptide and a test compound; and exposing thesample to the test compound and detecting a biological effect. Inpreferred embodiments, the RTP polypeptide is selected from the groupconsisting of RTP1-4 and RTP1-A1, RTP1-D1, and RTP1-D3. In preferredembodiments, the sample comprises a cell. In preferred embodiments, thesample comprises a tissue. In preferred embodiments, the sample is foundin a subject. In some embodiments, the biological effect comprises achange in activity of RTP. In some embodiments, the biological effectcomprises a change in expression of RTP.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a screening strategy for identifying molecules that promotecell-surface expression of odorant receptors. REEP1 was obtained fromDigital Differential Display analysis. RTP1 was obtained from SAGElibraries.

FIG. 2 shows REEP and/or RTP promote cell-surface expression of odorantreceptors in 293T Cells. (A) cDNAs encoding diverse ORs (MOR203-1, OREG,olfr62, OR-S46 and rat I7) were transfected with or without REEP1, RTP1and/or RTP2. Increased cell-surface staining of ORs was seen in cellsco-expressing the accessory proteins. In contrast, no difference incell-surface staining was seen in cells expressing β2 adrenergicreceptors. Using living-cell staining protocols, cell-surfacefluorescent signals are seen as distinctive punctate staining. Scale barequals to 50 um. (B) Normalized numbers of labelled cells is shown foreach transfection condition (N=4918-15526). After doubleimmunofluorescent staining against Rho-tagged receptors and theHA-tagged β2 adrenergic receptor, FACS analysis was performed toquantify immunopositive cells. The number of Rho-tagged receptorpositive cells was normalized to that of HA-tagged β2 adrenergicreceptor positive cells. In almost all cases, more immunopositive cellswere observed when different ORs were expressed with REEP1, RTP1 and/orRTP2. In contrast, when VR4 and mT2R5 receptor was used instead of ORs,no enhancement was observed. (C) Normalized mean fluorescence oflabelled cells is shown. The mean fluorescence of β2 adrenergic receptorwas used as a control. Stronger fluorescence was observed when differentORs were expressed with REEP1, RTP1 and/or RTP2. In contrast, when VR4and mT2R5 receptor was used instead of ORs, no enhancement was observed.(D) A summary of the FACS analysis is shown.

FIG. 3 shows that REEP and/or RTP do not promote cell-surface expressionof VR4 and mT2R5 in 293T Cells. cDNAs encoding VR4 and mT2R5 weretransfected with or without REEP1, RTP1 and/or RTP2. Unlike ORs,increased cell-surface staining was not seen in cells expressing theseproteins. BFP expression is shown to demonstrate high (˜70%)transfection efficiency of VR4 transfected cells. Using living-cellstaining protocols, cell-surface fluorescent signals are seen asdistinctive punctate staining. Scale bar equals to 50 um.

FIG. 4 presents fluorescent histogram data for REEP1, RTP1, and RTP2expression with odorant receptor (A) olfr62 and (B) mT2R5.

FIG. 5 shows the REEP and the RTP families. (A) Deduced amino acidsequences of REEP1(SEQ ID NO: 21). Solid bar indicates putativetransmembrane region (TM). The first TM region could function as asignal peptide. (B) Unrooted phylogenetic tree of REEP family members.At least 6 REEP family members (REEP1-6) were identified on the mousegenome. Yeast YOP1P, barley HVA22, and human DPI are homologous to REEPproteins. (C) Deduced amino acid sequences of RTP1 (SEQ ID NO: 33) andRTP2(SEQ ID NO: 34). Solid bar indicates putative transmembrane domain.Shaded amino acids are conserved between RTP1 and RTP2. There are twomore members (RTP3 and 4) on the mouse genome.

FIG. 6 shows expression of REEP1, RTP1 and RTP2. (A) Northern blotanalysis. Total RNA was used for northern blotting analysis. Olfactoryepithelium, vomeronasal organ, and brain showed ˜3.6 kb bandscorresponding to REEP1 mRNA. Only olfactory epithelium and vomeronasalorgan RNAs showed ˜3.5 kb and ˜2.6 kb bands corresponding RTP1 and RTP2mRNA, respectively. Ethidium bromide staining for 18S rRNA is shown as acontrol. (B) In situ hybridization analysis in the olfactory epithelium.Among REEP members, only REEP1 was expressed specifically by theolfactory neurons. REEP6 was expressed by supporting cells. Among RTPmembers, RTP1 and RTP2 are strongly expressed by the olfactory neurons.RTP4 was also expressed by the olfactory neurons but at much lowerlevel. OMP is a marker for mature olfactory neurons. Highermagnification of REEP1, RTP1, and RTP2 suggests that all olfactoryneurons may express all three molecules. Scale bar: 200 um (70 um inhigh magnification pictures). (C) In situ analysis of REEP1 in thebrain. REEP1 was expressed by a subset of brain cells. Scale bar: 200um.

FIG. 7 shows association of odorant Receptors with REEP1 and RTP1. (A)Control western blot analysis indicating expression of HA-taggedMOR203-1, Flag-tagged REEP1, RTP1 and ICAP1 in 293T cells. (B) WhenFlag-RTP1 or Flag-REEP1 was precipitated, HA-MOR203-1 proteins wereco-precipitated (Lanes 1 and 2). However, when Flag-ICAP-1 (a negativecontrol protein) was precipitated, HA-MOR203-1 proteins were notdetected (Lane 3). (C) When HA-MOR203-1 was precipitated, Flag-REEP1 andFlag-RTP1 were co-purified when co-expressed (Lanes 1 and 2). Negativecontrol protein (Flag-ICAP-1) was not co-precipitated (Lane 3).Asterisks indicate nonspecific Ig proteins. (D) Little cell-surfaceexpression was observed when RTP1 was transfected in 293T cells.However, when RTP1 and an odorant receptor (OREG) were co-transfected,more RTP1 staining signal was observed. (E) A small amount ofcell-surface signal was observed when REEP1 was transfected in 293Tcells. Co expression of an OR (olfr62) did not change the expression ofREEP1. Scale bars equal to 50 um.

FIG. 8 shows that expression of REEP1, RTP1 or RTP 2 enhances odorantreceptor activation. (A) Diagram showing cAMP responsive element (CRE)and luciferase was used to monitor activation of ORs. Activation of ORsincreases cAMP, which enhances the expression of luciferase reportergene through the CRE. (B) Normalized luciferase activities±SEM (N=4).REEP1, RTP1 and RTP2, expressed in various combination together withOREG, enhanced luciferase activities compared to OR alone. (C) Relativeluciferase activities+SEM (N=4). OREG or OR-S46 was used to ask ifREEP1, RTP1, or RTP2 could change ligand specificities of ORs. To obtainrelative activation to different odorants, luciferase activity to 300 uMof vanillin (OREG) or decanoic acid (OR-S46) was regarded as 1 in eachexpression condition. (D) Normalized luciferase activities+SEM (N=8).Enhanced response in Hana3A cells, a stable cell line expressing REEP1,RTP1, RTP2 and Golf, when three different ORs were expressed. (E) cAMPassays. Enhanced cAMP production to various concentrations of eugenol inHana3A cells when OREG was transfected. In contrast, cAMP production wasnot different between Hana3A cells and 293T cells expressing G_(αolf)when β2 adrenergic receptor was transfected and isoproterenol was used.

FIG. 9 shows RT-PCR analysis of Hana3A cells; + indicates PCR productsusing cDNA samples from Hana3A cells as template DNA; − indicatesnegative controls without reverse transcriptase; M indicates DNA marker.

FIG. 10 shows cell-surface expression of odorant receptors in Hana3A and293T cells. cDNAs encoding three ORs (OREG, olfr62 and OR-S46) weretransfected into Hana3A cells or 293T cells. Increased cell-surfacestaining was seen in Hana3A cells. Scale bar equals to 50 um.

FIG. 11 shows recognition profiles of odorant receptors to odorants. (A)Test odorants are shown on the left. The color indicate relativeluciferase activities (N=4). Each OR responded to different subset ofodorants. (B) and (C) Normalized luciferase activities (N=4). 139chemicals were used for initial ligand screening of MOR203-1 and olfr62.MOR203-1 responded to nonanoic acid. Olfr62 responded to five relatedaromatic compounds.

FIG. 12 shows cell-surface expression of 8 odorant receptors in Hana3Acells. Scale bar equals to 50 um.

FIG. 13 shows models for the roles of REEP and/or RTP in odorantreceptor expression.

FIG. 14 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 1) and aminoacid sequence (SEQ ID NO: 21) for murine REEP1.

FIG. 15 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 2) and aminoacid sequence (SEQ ID NO: 22) for murine REEP2.

FIG. 16 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 3) and aminoacid sequence (SEQ ID NO: 23) for murine REEP3.

FIG. 17 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 4) and aminoacid sequence (SEQ ID NO: 24) for murine REEP4.

FIG. 18 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 5) and aminoacid sequence (SEQ ID NO: 25) for murine REEP5.

FIG. 19 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 6) and aminoacid sequence (SEQ ID NO: 26) for murine REEP6.

FIG. 20 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 7) and aminoacid sequence (SEQ ID NO: 27) for human REEP1.

FIG. 21 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 8) and aminoacid sequence (SEQ ID NO: 28) for human REEP2.

FIG. 22 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 9) and aminoacid sequence (SEQ ID NO: 29) for human REEP3.

FIG. 23 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 10) and aminoacid sequence (SEQ ID NO: 30) for human REEP4.

FIG. 24 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 11) and aminoacid sequence (SEQ ID NO: 31) for human REEP5.

FIG. 25 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 12) and aminoacid sequence (SEQ ID NO: 32) for human REEP6.

FIG. 26 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 13) and aminoacid sequence (SEQ ID NO: 33) for murine RTP1.

FIG. 27 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 14) and aminoacid sequence (SEQ ID NO: 34) for murine RTP2.

FIG. 28 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 15) and aminoacid sequence (SEQ ID NO: 35) for murine RTP3.

FIG. 29 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 16) and aminoacid sequence (SEQ ID NO: 36) for murine RTP4.

FIG. 30 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 17) and aminoacid sequence (SEQ ID NO: 37) for human RTP1.

FIG. 31 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 18) and aminoacid sequence (SEQ ID NO: 38) for human RTP2.

FIG. 32 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 19) and aminoacid sequence (SEQ ID NO: 39) for human RTP3.

FIG. 33 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 20) and aminoacid sequence (SEQ ID NO: 40) for human RTP4.

FIG. 34 shows the activation patterns of human odorant receptors inresponse to odiferous agent exposure.

FIG. 35 schematically shows amino acid segments of RTP1-A, RTP1-B,RTP1-C, RTP1-D, and RTP1-E in comparison to RTP1.

FIG. 36 shows the murine amino acid sequence for RTP1-A (SEQ ID NO: 41).

FIG. 37 shows the murine amino acid sequence for RTP1-B (SEQ ID NO: 42).

FIG. 38 shows the murine amino acid sequence for RTP1-C (SEQ ID NO: 43).

FIG. 39 shows the murine amino acid sequence for RTP1-D (SEQ ID NO: 44).

FIG. 40 shows the murine amino acid sequence for RTP1-E (SEQ ID NO: 45).

FIG. 41 shows cell-surface expression of OLFR62 in Hana3A and 293Tcells. cDNAs encoding RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-Ewere transfected into Hana3A cells or 293T cells. Increased cell-surfacestaining was seen in Hana3A cells and 239T cells expressing RTP1-D.

FIG. 42 schematically shows a luciferase assay used to monitor theactivity of OLFR62 activity. cAMP responsive element (CRE) andluciferase was used to monitor activation of OLFR62. Activation ofOLFR62 increases cAMP, which enhances the expression of luciferasereporter gene through the CRE.

FIG. 43 shows OLFR62 activity as indicated by luciferase expression inHana3A cells and 293T cells expressing RTP1, RTP1-A, RTP1-B, RTP1-C,RTP1-D, RTP1-E, and control pCI.

FIG. 44 schematically shows the amino acid segments of RTP1-A1, RTP1-D1,RTP1-D2, and RTP1-D3 in comparison to RTP1-A and RTP1-D, respectively.

FIG. 45 shows the murine amino acid sequence for RTP1-A1 (SEQ ID NO:46), and the human amino acid sequence for RTP1-A1 (SEQ ID NO: 47).

FIG. 46 shows the murine amino acid sequence for RTP1-D1 (SEQ ID NO:48).

FIG. 47 shows the murine amino acid sequence for RTP-D2 (SEQ ID NO: 49).

FIG. 48 shows the murine amino acid sequence for RTP-D3 (SEQ ID NO: 50).

FIG. 49 shows cell-surface expression of OLFR62 in 293T cells. cDNAsencoding RTP1, RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, and control pCIwere transfected into 293T cells. Increased cell-surface staining wasseen in 239T cells expressing RTP1-A1, RTP1-D1 and RTP1-D3.

FIG. 50 shows OLFR62, OREG, S6, and 23-1 activity as indicated byluciferase expression in 293T cells expressing RTP1, RTP1-A1, RTP1-D1,RTP1-D2, and RTP1-D3, and control pCI.

FIG. 51 shows OLFR62, OREG, S6, and 23-1 activity as indicated byluciferase expression in Hana3A cells expressing RTP1, RTP1-A1, RTP1-D1,RTP1-D2, and RTP1-D3, and control pCI.

FIG. 52 shows cell-surface expression of OLFR62, OREG, MOR203-1, S6, and23-1 in 293T cells co-transfected with either RTP1, RTP1-A1 or controlpCI. cDNAs encoding RTP1, RTP1-A1, and control pCI were transfected intocells.

FIG. 53 schematically shows the amino acid segments of RTP1-A1-A(Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A(Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6).

FIG. 54 shows cell-surface expression of an OR in cells expressing RTP1,RTP4, Chimera 1, Chimera 2, Chimera 3, Chimera 4, Chimera 5, Chimera 6,and control pCI. cDNAs encoding RTP1, RTP4, RTP1-A1, Chimera 1, Chimera2, Chimera 3, Chimera 4, Chimera 5, Chimera 6, and control pCI weretransfected into 293T cells.

FIG. 55 shows OLFR62, OREG, S6, and 23-1 activity as indicated byluciferase expression in 293T cells expressing RTP1, RTP4, RTP1-A1,RTP1-D1, RTP1-D2, Chimera 1, Chimera 2, Chimera 3, Chimera 4, Chimera 5,Chimera 6, and control pCI.

FIG. 56 shows detection of RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-A1,RTP1-D, Chimera 4, Chimera 5, RTP1-D3, RTP1-D1, Chimera 6, and RTP4using anti-RTP1.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “REEP” when used in reference to proteins ornucleic acid refers to a REEP protein or nucleic acid encoding a REEPprotein of the present invention. The term REEP encompasses bothproteins that are identical to wild-type REEPs (e.g., REEP1, REEP2,REEP3, REEP4, REEP5, and REEP6) and those that are derived fromwild-type REEP (e.g. variants of REEP polypeptides of the presentinvention). In some embodiments, the “REEP” is a wild type murine REEPnucleic acid (mRNA) (e.g., SEQ ID NOs: 1-6) or a polypeptide encoded bythe wild type murine REEP amino acid sequence (e.g., SEQ ID NOs:21-26).In other embodiments, the “REEP” is a wild type human REEP nucleic acid(mRNA) (e.g., SEQ ID NOs: 7-12) or a polypeptide encoded by a wild typehuman REEP amino acid sequence (e.g., SEQ ID NOs: 21-32). Examples ofREEP proteins or nucleic acids include, but are not limited to, REEP1,REEP2, REEP3, REEP4, REEP5 and REEP6.

As used herein, the term “RTP” when used in reference to proteins ornucleic acid refers to a RTP protein or nucleic acid encoding a RTPprotein of the present invention. The term RTP encompasses both proteinsthat are identical to wild-type RTPs (e.g., RTP1, RTP2, RTP3, and RTP4)and those that are derived from wild-type RTP (e.g. variants of RTPpolypeptides of the present invention including but not limited toRTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,RTP-D3, or chimeric genes constructed with portions of RTP1 codingregions (e.g., RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1(Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), andRTP4-A1-D1 (Chimera 6)). In some embodiments, the “RTP” is a wild typemurine RTP nucleic acid (mRNA) (e.g., SEQ ID NOs: 13-16) or apolypeptide encoded by the wild type or variant murine RTP amino acidsequence (e.g., SEQ ID NOs: 33-36, 41-50). In other embodiments, the“RTP” is a wild type human RTP nucleic acid (mRNA) (e.g., SEQ ID NOs:17-20) or a polypeptide encoded by a wild type human RTP amino acidsequence (e.g., SEQ ID NOs: 37-40). Examples of RTP proteins or nucleicacids include, but are not limited to, RTP1, RTP2, RTP3, RTP4, RTP1-A,RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3,RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3),RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera6).

As used herein, the term “odorant receptor” refers to odorant receptorsgenerated from olfactory sensory neurons. Examples of odorant receptorsinclude, but are not limited to, S6/79, S18, S46, S50, MOR23-1, MOR31-4,MOR31-6, MOR32-5 and MOR32-11.

As used herein, the term “odorant receptor cell surface localization” orequivalent terms refer to the molecular transport of an odorant receptorto a cell surface membrane. Examples of cell surface localizationincludes, but is not limited to, localization to cilia at the tip of adendrite, and localization to an axon terminal.

As used herein, the term “odorant receptor functional expression” orequivalent terms, refer to an odorant receptor's ability to interactwith an odorant receptor ligand (e.g., an odiferous molecule).

As used herein, the term “olfactory disorder,” “olfactory dysfunction,”“olfactory disease” or similar term refers to a disorder, dysfunction ordisease resulting in a diminished olfactory sensation (e.g., smellaberration). Examples of olfactory disorders, dysfunctions and/ordiseases include, but are not limited to, head trauma, upper respiratoryinfections, tumors of the anterior cranial fossa, Kallmann syndrome,Foster Kennedy syndrome, Parkinson's disease, Alzheimer's disease,Huntington chorea, and exposure to toxic chemicals or infections.Diminished olfactory sensation is classified as anosmia—absence of smellsensation; hyposmia—decreased smell sensation; dysosmia—distortion ofsmell sensation; cacosmia—sensation of a bad or foul smell; andparosmia—sensation of smell in the absence of appropriate stimulus.

As used herein, the term “REEP1” when used in reference to a protein ornucleic acid refers to a REEP1 protein or nucleic acid encoding a REEP1protein of the present invention. The term REEP1 encompasses bothproteins that are identical to wild-type REEP1 and those that arederived from wild type REEP1 (e.g., variants of REEP1 polypeptides ofthe present invention) or chimeric genes constructed with portions ofREEP1 coding regions). In some embodiments, the “REEP1” is a wild typemurine REEP1 nucleic acid (mRNA) (SEQ ID NO: 1) or polypeptide encodedby the wild type murine amino acid sequence (SEQ ID NO: 21). In otherembodiments, the “REEP1” is a wild type human REEP1 nucleic acid (mRNA)(SEQ ID NO: 7) or polypeptide encoded by the wild type human REEP1 aminoacid sequence (SEQ ID NO: 27). In other embodiments, the “REEP1” is avariant or mutant nucleic acid or amino acid.

As used herein, the term “RTP1” when used in reference to a protein ornucleic acid refers to a RTP1 protein or nucleic acid encoding a RTP1protein of the present invention. The term RTP1 encompasses bothproteins that are identical to wild-type RTP1 and those that are derivedfrom wild type RTP1 (e.g., variants of RTP1 polypeptides of the presentinvention including but not limited to RTP1-A, RTP1-B, RTP1-C, RTP1-D,RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3) or chimeric genes constructedwith portions of RTP1 coding regions (e.g., RTP1-A1-A (Chimera 1),RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4),RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6)). In someembodiments, the “RTP1” is a wild type murine RTP1 nucleic acid (mRNA)(SEQ ID NO: 13) or polypeptide encoded by the wild type murine aminoacid sequence (SEQ ID NO: 33). In other embodiments, the “RTP1” is awild type human RTP1 nucleic acid (mRNA) (SEQ. ID NO: 17) or polypeptideencoded by the wild type human RTP1 amino acid sequence (SEQ ID NO: 37).In other embodiments, the “RTP1” is a variant or mutant nucleic acid oramino acid.

As used herein, the term “RTP2” when used in reference to a protein ornucleic acid refers to a RTP2 protein or nucleic acid encoding a RTP2protein of the present invention. The term RTP2 encompasses bothproteins that are identical to wild-type RTP2 and those that are derivedfrom wild type RTP2 (e.g., variants of RTP2 polypeptides of the presentinvention) or chimeric genes constructed with portions of RTP2 codingregions). In some embodiments, the “RTP2” is a wild type murine RTP2nucleic acid (mRNA) (SEQ ID NO: 14) or polypeptide encoded by the wildtype murine amino acid sequence (SEQ ID NO: 34). In other embodiments,the “RTP2” is a wild type human RTP2 nucleic acid (mRNA) (SEQ ID NO: 18)or polypeptide encoded by the wild type human REEP1 amino acid sequence(SEQ ID NO: 38). In other embodiments, the “RTP2” is a variant or mutantnucleic acid or amino acid.

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal like a dog, cat, bird, livestock, and preferably ahuman. Specific examples of “subjects” and “patients” include, but arenot limited to, individuals with an olfactory disorder, and individualswith olfactory disorder-related characteristics or symptoms.

As used herein, the phrase “symptoms of an olfactory disorder” and“characteristics of an olfactory disorder” include, but are not limitedto, a diminished olfactory sensation (e.g., smell aberration).

The phrase “under conditions such that the symptoms are reduced” refersto any degree of qualitative or quantitative reduction in detectablesymptoms of olfactory disorders, including but not limited to, adetectable impact on the rate of recovery from disease, or the reductionof at least one symptom of an olfactory disorder.

The term “siRNAs” refers to short interfering RNAs. Methods for the useof siRNAs are described in U.S. Patent App. No.: 20030148519/A1 (hereinincorporated by reference). In some embodiments, siRNAs comprise aduplex, or double-stranded region, of about 18-25 nucleotides long;often siRNAs contain from about two to four unpaired nucleotides at the3′ end of each strand. At least one strand of the duplex ordouble-stranded region of a siRNA is substantially homologous to orsubstantially complementary to a target RNA molecule. The strandcomplementary to a target RNA molecule is the “antisense strand;” thestrand homologous to the target RNA molecule is the “sense strand,” andis also complementary to the siRNA antisense strand. siRNAs may alsocontain additional sequences; non-limiting examples of such sequencesinclude linking sequences, or loops, as well as stem and other foldedstructures. siRNAs appear to function as key intermediaries intriggering RNA interference in invertebrates and in vertebrates, and intriggering sequence-specific RNA degradation during posttranscriptionalgene silencing in plants.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi may also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the terms “instructions for using said kit for saiddetecting the presence or absence of a variant REEP1 nucleic acid orpolypeptide in said biological sample,” “instructions for using said kitfor said detecting the presence or absence of a variant RTP1 nucleicacid or polypeptide in said biological sample,” “instructions for usingsaid kit for said detecting the presence or absence of a variant RTP2nucleic acid or polypeptide in said biological sample” includeinstructions for using the reagents contained in the kit for thedetection of variant and wild type REEP and/or RTP nucleic acids orpolypeptides.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, RNA (e.g., including but not limited to, mRNA, tRNA andrRNA) or precursor (e.g., REEP1, RTP1 or RTP2). The polypeptide, RNA, orprecursor can be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb on either end such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene that are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide.

In particular, the term “REEP1 gene,” “RTP1 gene,” “RTP1 genes,” “RTP2gene,” or “RTP2 genes” refer to the full-length respective REEP and/orRTP nucleotide sequence (e.g., contained in SEQ ID NOs:1, 2 and 3).However, it is also intended that the term encompass fragments of theREEP and/or RTP sequences (e.g., RTP1-A, RTP1-B, RTP1-C, RTP1-D, andRTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3), chimeric genes constructedwith portions of RTP1 coding regions (e.g., RTP1-A1-A (Chimera 1),RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4),RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6)), mutants of the REEPand/or RTP sequences, as well as other domains within the full-lengthREEP and/or RTP nucleotide sequences. Furthermore, the terms “REEP1nucleotide sequence,” “REEP1 polynucleotide sequence,” “RTP1 nucleotidesequence,” “RTP1 polynucleotide sequence,” “RTP2 nucleotide sequence,”or “RTP2 polynucleotide sequence” encompasses DNA sequences, cDNAsequences, RNA (e.g., mRNA) sequences, and associated regulatorysequences.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified,” “mutant,” “polymorphism,” and “variant” refer to a gene orgene product that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotides orpolynucleotide, referred to as the “5′ end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements that direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or, in other words, the nucleic acid sequencethat encodes a gene product. The coding region may be present in a cDNA,genomic DNA, or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence5′-“A-G-T-3′,” is complementary to the sequence 3′-“T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids. Complementarity can include the formationof base pairs between any type of nucleotides, including non-naturalbases, modified bases, synthetic bases and the like.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to inhibition of binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “competes for binding” is used in reference toa first polypeptide with an activity which binds to the same substrateas does a second polypeptide with an activity, where the secondpolypeptide is a variant of the first polypeptide or a related ordissimilar polypeptide. The efficiency (e.g., kinetics orthermodynamics) of binding by the first polypeptide may be the same asor greater than or less than the efficiency substrate binding by thesecond polypeptide. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twopolypeptides. The term “K_(m)” as used herein refers to theMichaelis-Menton constant for an enzyme and is defined as theconcentration of the specific substrate at which a given enzyme yieldsone-half its maximum velocity in an enzyme catalyzed reaction.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The present invention is not limited to the hybridization of probes ofabout 500 nucleotides in length. The present invention contemplates theuse of probes between approximately 10 nucleotides up to severalthousand (e.g., at least 5000) nucleotides in length. One skilled in therelevant understands that stringency conditions may be altered forprobes of other sizes (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985] and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY[1989]).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman [Smithand Waterman, Adv. Appl. Math. 2: 482 (1981)] by the homology alignmentalgorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol.48:443 (1970)], by the search for similarity method of Pearson andLipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.) 85:2444(1988)], by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention (e.g., REEP1, RTP1 orRTP2).

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions (claimed in thepresent invention) with its various ligands and/or substrates.

The term “polymorphic locus” is a locus present in a population thatshows variation between members of the population (i.e., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

As used herein, the term “genetic variation information” or “geneticvariant information” refers to the presence or absence of one or morevariant nucleic acid sequences (e.g., polymorphism or mutations) in agiven allele of a particular gene (e.g., a REEP and/or RTP gene of thepresent invention).

As used herein, the term “detection assay” refers to an assay fordetecting the presence or absence of variant nucleic acid sequences(e.g., polymorphisms or mutations) in a given allele of a particulargene (e.g., a REEP and/or RTP gene).

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” refers to a nucleic acid sequence orstructure to be detected or characterized. Thus, the “target” is soughtto be sorted out from other nucleic acid sequences. A “segment” isdefined as a region of nucleic acid within the target sequence.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule that is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

As used herein, the term “antisense” is used in reference to RNAsequences that are complementary to a specific RNA sequence (e.g.,mRNA). Included within this definition are antisense RNA (“asRNA”)molecules involved in gene regulation by bacteria. Antisense RNA may beproduced by any method, including synthesis by splicing the gene(s) ofinterest in a reverse orientation to a viral promoter that permits thesynthesis of a coding strand. Once introduced into an embryo, thistranscribed strand combines with natural mRNA produced by the embryo toform duplexes. These duplexes then block either the furthertranscription of the mRNA or its translation. In this manner, mutantphenotypes may be generated. The term “antisense strand” is used inreference to a nucleic acid strand that is complementary to the “sense”strand. The designation (−) (i.e., “negative”) is sometimes used inreference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding REEP and/or RTP includes, by way ofexample, such nucleic acid in cells ordinarily expressing REEP and/orRTP where the nucleic acid is in a chromosomal location different fromthat of natural cells, or is otherwise flanked by a different nucleicacid sequence than that found in nature. The isolated nucleic acid,oligonucleotide, or polynucleotide may be present in single-stranded ordouble-stranded form. When an isolated nucleic acid, oligonucleotide orpolynucleotide is to be utilized to express a protein, theoligonucleotide or polynucleotide will contain at a minimum the sense orcoding strand (i.e., the oligonucleotide or polynucleotide maysingle-stranded), but may contain both the sense and anti-sense strands(i.e., the oligonucleotide or polynucleotide may be double-stranded).

As used herein, a “portion of a chromosome” refers to a discrete sectionof the chromosome. Chromosomes are divided into sites or sections bycytogeneticists as follows: the short (relative to the centromere) armof a chromosome is termed the “p” arm; the long arm is termed the “q”arm. Each arm is then divided into 2 regions termed region 1 and region2 (region 1 is closest to the centromere). Each region is furtherdivided into bands. The bands may be further divided into sub-bands. Forexample, the 11p15.5 portion of human chromosome 11 is the portionlocated on chromosome 11 (11) on the short arm (p) in the first region(1) in the 5th band (5) in sub-band 5 (0.5). A portion of a chromosomemay be “altered;” for instance the entire portion may be absent due to adeletion or may be rearranged (e.g., inversions, translocations,expanded or contracted due to changes in repeat regions). In the case ofa deletion, an attempt to hybridize (i.e., specifically bind) a probehomologous to a particular portion of a chromosome could result in anegative result (i.e., the probe could not bind to the sample containinggenetic material suspected of containing the missing portion of thechromosome). Thus, hybridization of a probe homologous to a particularportion of a chromosome may be used to detect alterations in a portionof a chromosome.

The term “sequences associated with a chromosome” means preparations ofchromosomes (e.g., spreads of metaphase chromosomes), nucleic acidextracted from a sample containing chromosomal DNA (e.g., preparationsof genomic DNA); the RNA that is produced by transcription of geneslocated on a chromosome (e.g., hnRNA and mRNA), and cDNA copies of theRNA transcribed from the DNA located on a chromosome. Sequencesassociated with a chromosome may be detected by numerous techniquesincluding probing of Southern and Northern blots and in situhybridization to RNA, DNA, or metaphase chromosomes with probescontaining sequences homologous to the nucleic acids in the above listedpreparations.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets, whichspecify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, REEP and/or RTP antibodiesare purified by removal of contaminating non-immunoglobulin proteins;they are also purified by the removal of immunoglobulin that does notbind a REEP and/or RTP polypeptide. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind a REEPand/or RTP polypeptide results in an increase in the percent of REEP1,RTP1 or RTP2-reactive immunoglobulins in the sample. In another example,recombinant REEP and/or RTP polypeptides are expressed in bacterial hostcells and the polypeptides are purified by the removal of host cellproteins; the percent of recombinant REEP and/or RTP polypeptides isthereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein, is used to indicate a proteinthat does not contain amino acid residues encoded by vector sequences;that is the native protein contains only those amino acids found in theprotein as it occurs in nature. A native protein may be produced byrecombinant means or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 [1989]).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The term “transgene” as used herein refers to a foreign, heterologous,or autologous gene that is placed into an organism by introducing thegene into newly fertilized eggs or early embryos. The term “foreigngene” refers to any nucleic acid (e.g., gene sequence) that isintroduced into the genome of an animal by experimental manipulationsand may include gene sequences found in that animal so long as theintroduced gene does not reside in the same location as does thenaturally-occurring gene. The term “autologous gene” is intended toencompass variants (e.g., polymorphisms or mutants) of the naturallyoccurring gene. The term transgene thus encompasses the replacement ofthe naturally occurring gene with a variant form of the gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis (See,Example 10, for a protocol for performing Northern blot analysis).

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodingREEP1s, RTP1s or RTP2s (e.g., SEQ ID NOs:1, 2 and 3) or fragmentsthereof may be employed as hybridization probes. In this case, the REEPand/or RTP encoding polynucleotide sequences are typically employed inan aqueous solution containing salts (e.g., NaCl), detergents (e.g.,SDS), and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

As used herein, the term “response,” when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

As used herein, the term “reporter gene” refers to a gene encoding aprotein that may be assayed. Examples of reporter genes include, but arenot limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol.7:725 [1987] and U.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from CLONTECH Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, β-galactosidase,alkaline phosphatase, and horse radish peroxidase.

General Description

Continued progress in understanding olfactory coding has beensignificantly hampered by the inability to functionally express ORs inheterologous cells in order to identify cognate ligands. To overcomethis problem, experiments conducted during the course of the presentinvention searched for molecules that are included in cell-surfaceexpression of ORs. Three transmembrane proteins, REEP1, RTP1, and RTP2,as well as variants thereof, were identified that promote functionalcell surface expression of ORs in 293T cells. REEP and/or RTP areexpressed specifically by olfactory neurons in the olfactory epithelium.REEP1 and RTP1 interacts with OR proteins. Using cells expressing REEP1and RTP1 and RTP2, new ORs that respond to aliphatic odorants wereidentified. The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, experiments conductedduring the course of the present invention demonstrated the importanceof the accessory proteins of ORs in functional cell-surface expressionand in decoding OR-ligand specificities.

The identification and use of proteins involved in the localization ofORs provides numerous research, diagnostic, drug screening, andtherapeutic applications. For example, the nucleic acids and proteins ofthe present invention permit the selective and controllable presentationof ORs on test cells to, among other things, identify new ORs,characterize ORs, identify OR ligands, correlate olfactory responses tothe molecular interactions underlying such response, identify andcharacterize groups of ORs and ligands responsible for olfactoryresponses and health conditions, and identify, select, and characterizeregulators of OR response to study and control olfactory responses. Thepresent invention, also, thus provides means for manipulating olfactoryresponses and the molecular basis for such response in vitro and invivo. Numerous commercial applications are thus made possible, includingthe production, characterization, and use of in vitro or in vivo cellarrays expressing desired localized ORs for screening (e.g.,high-throughput screening) compounds or use as synthetic olfactorysystems. Any industry, including food industries, health industries,cosmetic industries, militaries, sanitary agencies, animal sniffers(e.g., for drugs, explosives, accident victims, etc.), among many otherswill find use of the compositions and methods of the present invention.

Inhibitors (e.g., antibodies, small molecules, aptamers, etc.) ofOR/ligand interactions that are identified by the methods of the presentinvention find may uses. For example, the present invention provides asystematic way to identify which receptors and ligands are responsiblefor particular olfactory sensations (e.g., perceived scents). Thus, forexample, by blocking particular interactions (e.g., via a nasal sprayhaving the inhibitors) or enhancing particular interactions (e.g., via anasal spray that provides certain ligands or a coating on the surface ofan object that emits certain ligands) one can control perceived scents.Thus, undesired scents can be blocked, covered, or altered (e.g., asniffer dog can be treated so as to only smell a target of interestedand no other distracting smells, a sanitary worked can be made immune tothe scent of waste, etc.) and desired scents can be enhanced.

The present invention also provides novel gene and protein sequences andmethods of their use. A detailed description of certain preferredembodiments and uses of the present invention is described below. Thepresent invention is not limited to these particular illustrativeembodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to peptides capable of promoting odorantreceptor cell-surface localization and odorant receptor functionalexpression. The present invention further provides assays for thedetection of therapeutic agents, and for the detection of odorantreceptor accessory protein polymorphisms and mutations associated withdisease states. Exemplary embodiments of the present invention aredescribed below.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. OlfactorySensation; II. REEP and RTP Polynucleotides; III. REEP and RTPPolypeptides; IV. Detection of REEP and RTP Alleles; V. Generation ofREEP and RTP Antibodies; VI. Gene Therapy Using REEP and RTP; VII.Transgenic Animals Expressing Exogenous REEP and RTP Genes and Homologs,Mutants, and Variants Thereof; VIII. Drug Screening Using REEP and RTP;IX. Pharmaceutical Compositions Containing REEP and RTP Nucleic Acid,Peptides, and Analogs; X. RNA Interference (RNAi); XI. RNAi for REEP andRTP; and XII. Identification of Odorant Receptor Ligands.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular cloning: a laboratory manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed., 1984); “Animalcell culture” (R. I. Freshney, ed., 1987); the series “Methods inenzymology” (Academic Press, Inc.); “Handbook of experimentalimmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene transfer vectorsfor mammalian cells” (J. M. Miller & M. P. Calos, eds., 1987); “Currentprotocols in molecular biology” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: the polymerase chain reaction” (Mullis et al.,eds., 1994); and “Current protocols in immunology” (J. E. Coligan etal., eds., 1991), each of which is herein incorporated by reference inits entirety.

I. Olfactory Sensation

The olfactory system represents one of the oldest sensory modalities inthe phylogenetic history of mammals. Olfaction is less developed inhumans than in other mammals such as rodents. As a chemical sensor, theolfactory system detects food and influences social and sexual behavior.The specialized olfactory epithelial cells characterize the only groupof neurons capable of regeneration. Activation occurs when odiferousmolecules come in contact with specialized processes known as theolfactory vesicles. Within the nasal cavity, the turbinates or nasalconchae serve to direct the inspired air toward the olfactory epitheliumin the upper posterior region. This area (only a few centimeters wide)contains more than 100 million olfactory receptor cells. Thesespecialized epithelial cells give rise to the olfactory vesiclescontaining kinocilia, which serve as sites of stimulus transduction.

There are three specialized neural systems are present within the nasalcavities in humans: 1) the main olfactory system (cranial nerve I), 2)trigeminal somatosensory system (cranial nerve V), 3) the nervusterminals (cranial nerve 0). CN I mediates odor sensation. It isresponsible for determining flavors. CN V mediates somatosensorysensations, including burning, cooling, irritation, and tickling. CN 0is a ganglionated neural plexus. It spans much of the nasal mucosabefore coursing through the cribriform plate to enter the forebrainmedial to the olfactory tract. The exact function of the nervusterminals is unknown in humans.

The olfactory neuroepithelium is a pseudostratified columnar epithelium.The specialized olfactory epithelial cells are the only group of neuronscapable of regeneration. The olfactory epithelium is situated in thesuperior aspect of each nostril, including cribriform plate, superiorturbinate, superior septum, and sections of the middle turbinate. Itharbors sensory receptors of the main olfactory system and some CN Vfree nerve endings. The olfactory epithelium loses its generalhomogeneity postnatally, and as early as the first few weeks of lifemetaplastic islands of respiratory-like epithelium appear. Themetaplasia increases in extent throughout life. It is presumed that thisprocess is the result of insults from the environment, such as viruses,bacteria, and toxins.

There are 6 distinct cells types in the olfactory neuroepithelium: 1)bipolar sensory receptor neurons, 2) microvillar cells, 3) supportingcells, 4) globose basal cells, 5) horizontal basal cells, 6) cellslining the Bowman's glands. There are approximately 6,000,000 bipolarneurons in the adult olfactory neuroepithelium. They are thin dendriticcells with rods containing cilia at one end and long central processesat the other end forming olfactory fila. The olfactory receptors arelocated on the ciliated dendritic ends. The unmyelinated axons coalesceinto 40 bundles, termed olfactory fila, which are ensheathed bySchwann-like cells. The fila transverses the cribriform plate to enterthe anterior cranial fossa and constitute CN I. Microvillar cells arenear the surface of the neuroepithelium, but the exact functions ofthese cells are unknown. Supporting cells are also at the surface of theepithelium. They join tightly with neurons and microvillar cells. Theyalso project microvilli into the mucus. Their functions includeinsulating receptor cells from one another, regulating the compositionof the mucus, deactivating odorants, and protecting the epithelium fromforeign agents. The basal cells are located near the basement membrane,and are the progenitor cells from which the other cell types arise. TheBowman's glands are a major source of mucus within the region of theolfactory epithelium.

The odorant receptors are located on the cilia of the receptor cells.Each receptor cell expresses a single odorant receptor gene. There areapproximately 1,000 classes of receptors at present. The olfactoryreceptors are linked to the stimulatory guanine nucleotide bindingprotein Golf. When stimulated, it can activate adenylate cyclase toproduce the second messenger cAMP, and subsequent events lead todepolarization of the cell membrane and signal propagation. Althougheach receptor cell only expresses one type of receptor, each cell iselectrophysiologically responsive to a wide but circumscribed range ofstimuli. This implies that a single receptor accepts a range ofmolecular entities.

The olfactory bulb is located on top of the cribriform plate at the baseof the frontal lobe in the anterior cranial fossa. It receives thousandsof primary axons from olfactory receptor neurons. Within the olfactorybulb, these axons synapse with a much smaller number of second orderneurons which form the olfactory tract and project to olfactory cortex.The olfactory cortex includes the frontal and temporal lobes, thalamus,and hypothalamus.

Although mammalian ORs were identified over 10 years ago, little isknown about the selectivity of the different ORs for chemical stimuli,mainly because it has been difficult to express ORs on the cell surfaceof heterologous cells and assay their ligand-binding specificity (see,e.g., Mombaerts, P. (2004) Nat Rev Neurosci 5, 263-278; hereinincorporated by reference in its entirety). The reason is that ORproteins are retained in the ER and subsequently degraded in theproteosome (see, e.g., Lu, M., et al., (2003) Traffic 4, 416-433;McClintock, T. S., (1997) Brain Res Mol Brain Res 48, 270-278; eachherein incorporated by reference in their entireties). Despite thesedifficulties, extensive efforts have matched about 20 ORs with cognateligands with various degrees of certainty (see, e.g., Bozza, T., et al.,(2002) J Neurosci 22, 3033-3043; Gaillard, I., et al., (2002) Eur JNeurosci 15, 409-418; Hatt, H., et al., (1999) Cell Mol Biol 45,285-291; Kajiya, K., et al., (2001) J Neurosci 21, 6018-6025;Krautwurst, D., et al., (1998) Cell 95, 917-926; Malnic, B., et al.,(1999) Cell 96, 713-723; Raming, K., et al., (1993) Nature 361, 353-356;Spehr, M., et al., (2003) Science 299, 2054-2058; Touhara, K., et al.,(1999) Proc Natl Acad Sci USA 96, 4040-4045; Zhao, H., et al., (1998)Science 279, 237-242; each herein incorporated by reference in theirentirety). Adding the 20 N-terminal amino acids of rhodopsin (e.g.,Rho-tag) or a foreign signal peptide to the N-terminus facilitatessurface expression of some ORs in heterologous cells (see, e.g., Hatt,H., et al., (1999) Cell Mol Biol 45, 285-291; Krautwurst, D., et al.,(1998) Cell 95, 917-926; each herein incorporated in their entirety).However, for most ORs, modifications do not reliably promotecell-surface expression. For example, ODR-4, which is required forproper localization of chemosensory receptors in C. elegans, has a smalleffect on facilitating cell-surface expression of one rat OR, but notanother OR (see, e.g., Gimelbrant, A. A., et al., (2001) J Biol Chem276, 7285-7290; herein incorporated by reference). These findingsindicate that olfactory neurons have a selective molecular machinerythat promotes proper targeting of OR proteins to the cell surface, butno components of this machinery have been identified (see, e.g.,Gimelbrant, A. A., et al., (2001) J Biol Chem 276, 7285-7290;McClintock, T. S., and Sammeta, N. (2003) Neuroreport 14, 1547-1552;each herein incorporated by reference in their entirety).

For some GPCRs, accessory proteins are required for correct targeting tothe cell surface membrane (see, e.g., Brady, A. E., and Limbird, L. E.(2002) Cell Signal 14, 297-309; herein incorporated by reference in itsentirety). These proteins include NinaA for Drosophila Rhodopsin (see,e.g., Baker, E. K., et al., (1994) Embo J 13, 4886-4895; Shieh, B. H.,et al., (1989) Nature 338, 67-70; each herein incorporated by referencein their entirety), RanBP2 for mammalian cone opsin (see, e.g.,Ferreira, P. A., et al., (1996) Nature 383, 637-640; herein incorporatedby reference in its entirety), RAMPs for the mammalian calcitoninreceptor-like receptor (CRLR) (see, e.g., McLatchie, L. M., et al.,(1998) Nature 393, 333-339; herein incorporated by reference in itsentirety) and finally the M10 family of MHC class I proteins and beta 2microglobulin for V2R5, the putative mammalian pheromone receptors (see,e.g., Loconto, J., et al., (2003) Cell 112, 607-618; herein incorporatedby reference in its entirety). With the exception of NinaA and RanBP2,none of these accessory proteins share any sequence homology to witheach other; their only common feature is their association with themembrane.

The present invention provides novel proteins (e.g., REEP1, RTP1, RTP2,RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1(Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), andRTP4-A1-D1 (Chimera 6)) promoting OR cell surface localization and ORfunctional expression, and numerous compositions and methods related tothese findings.

II. REEP and RTP Polynucleotides

As described above, the present invention provides novel proteinspromoting odorant receptor cell surface localization and odorantreceptor functional expression. In particular, the present inventionprovides REEP genes and polypeptides (e.g., REEP1, REEP2, REEP3, REEP4,REEP5, and REEP6) and RTP genes and polypeptides (e.g., RTP1, RTP2,RTP3, RTP4, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1,RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5),and RTP4-A1-D1 (Chimera 6)). In preferred embodiments, REEP1, RTP1,RTP2, and variants of RTP1 (e.g., RTP1-A, RTP1-B, RTP1-C, RTP1-D, andRTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1),RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4),RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6) promote odorantreceptor cell surface localization and odorant receptor functionalexpression.

Accordingly, the present invention provides nucleic acids encoding REEPgenes, homologs, variants (e.g., polymorphisms and mutants), includingbut not limited to, those described in SEQ ID NOs: 1-12. The presentinvention provides nucleic acids encoding RTP genes, homologs, variants(e.g., polymorphisms and mutants), including but not limited to, thosedescribed in SEQ ID NOs: 13-20. Table 1 describes exemplary REEP and RTPgenes of the present invention. In some embodiments, the presentinvention provides polynucleotide sequences that are capable ofhybridizing to SEQ ID NOs: 1-20 under conditions of low to highstringency as long as the polynucleotide sequence capable of hybridizingencodes a protein that retains a biological activity of the naturallyoccurring REEP and/or RTP protein. In some embodiments, the protein thatretains a biological activity of a naturally occurring REEP and/or RTPis 70% homologous to the wild-type REEP and/or RTP, preferably 80%homologous to the wild-type REEP and/or RTP, more preferably 90%homologous to the wild-type REEP and/or RTP, and most preferably 95%homologous to wild-type the REEP and/or RTP. In preferred embodiments,hybridization conditions are based on the melting temperature (T_(m)) ofthe nucleic acid binding complex and confer a defined “stringency” asexplained above (see e.g., Wahl, et al., Meth. Enzymol., 152:399-407(1987), incorporated herein by reference).

In other embodiments of the present invention, additional alleles ofREEP and/or RTP genes are provided. In preferred embodiments, allelesresult from a polymorphism or mutation (i.e., a change in the nucleicacid sequence) and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one or many allelic forms. Common mutational changes that giverise to alleles are generally ascribed to deletions, additions orsubstitutions of nucleic acids. Each of these types of changes may occuralone, or in combination with the others, and at the rate of one or moretimes in a given sequence. Additional examples include truncationmutations (e.g., such that the encoded mRNA does not produce a completeprotein).

In still other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aREEP and/or RTP coding sequence for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns, to change codon preference, etc.). Variants of RTP1 includebut are not limited to RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E,RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2(Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2(Chimera 5), and RTP4-A1-D1 (Chimera 6).

In some embodiments of the present invention, the polynucleotidesequence of REEP and/or RTP may be extended utilizing the nucleotidesequence in various methods known in the art to detect upstreamsequences such as promoters and regulatory elements. For example, it iscontemplated that restriction-site polymerase chain reaction (PCR) willfind use in the present invention. This is a direct method that usesuniversal primers to retrieve unknown sequence adjacent to a known locus(Gobinda et al., PCR Methods Applic., 2:318-22 (1993); hereinincorporated by reference in its entirety). First, genomic DNA isamplified in the presence of a primer to a linker sequence and a primerspecific to the known region. The amplified sequences are then subjectedto a second round of PCR with the same linker primer and anotherspecific primer internal to the first one. Products of each round of PCRare transcribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

In another embodiment, inverse PCR can be used to amplify or extendsequences using divergent primers based on a known region (Triglia etal., Nucleic Acids Res., 16:8186 [1988]). The primers may be designedusing Oligo 4.0 (National Biosciences Inc, Plymouth Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68-72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template. In still other embodiments, walking PCR is utilized.Walking PCR is a method for targeted gene walking that permits retrievalof unknown sequence (Parker et al., Nucleic Acids Res., 19:3055-60[1991]). The PROMOTERFINDER kit (Clontech) uses PCR, nested primers andspecial libraries to “walk in” genomic DNA. This process avoids the needto screen libraries and is useful in finding intron/exon junctions.

Preferred libraries for screening for full length cDNAs includemammalian libraries that have been size-selected to include largercDNAs. Also, random primed libraries are preferred, in that they willcontain more sequences that contain the 5′ and upstream gene regions. Arandomly primed library may be particularly useful in case where anoligo d(T) library does not yield full-length cDNA. Genomic mammalianlibraries are useful for obtaining introns and extending 5′ sequence.

In other embodiments of the present invention, variants of the disclosedREEP and/or RTP sequences are provided. In preferred embodiments,variants result from polymorphisms or mutations (i.e., a change in thenucleic acid sequence) and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one, or many variant forms. Common mutationalchanges that give rise to variants are generally ascribed to deletions,additions or substitutions of nucleic acids. Each of these types ofchanges may occur alone, or in combination with the others, and at therate of one or more times in a given sequence.

It is contemplated that it is possible to modify the structure of apeptide having a function (e.g., REEP and/or RTP function) for suchpurposes as altering the biological activity (e.g., altered REEP and/orRTP function). Such modified peptides are considered functionalequivalents of peptides having an activity of a REEP and/or RTP peptideas defined herein. A modified peptide can be produced in which thenucleotide sequence encoding the polypeptide has been altered, such asby substitution, deletion, or addition. In particularly preferredembodiments, these modifications do not significantly reduce thebiological activity of the modified REEP and/or RTP genes. In otherwords, construct “X” can be evaluated in order to determine whether itis a member of the genus of modified or variant REEP and/or RTP of thepresent invention as defined functionally, rather than structurally. Inpreferred embodiments, the activity of variant REEP and/or RTPpolypeptides is evaluated by methods described herein (e.g., thegeneration of transgenic animals or the use of signaling assays).

Moreover, as described above, variant forms of REEP and/or RTP genes arealso contemplated as being equivalent to those peptides and DNAmolecules that are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of REEP and/or RTPcontaining conservative replacements. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids can be divided intofour families: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalpolypeptide can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

As described in more detail below, variants may be produced by methodssuch as directed evolution or other techniques for producingcombinatorial libraries of variants, described in more detail below. Instill other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aREEP and/or RTP coding sequence including, but not limited to,alterations that modify the cloning, processing, localization,secretion, and/or expression of the gene product. For example, mutationsmay be introduced using techniques that are well known in the art (e.g.,site-directed mutagenesis to insert new restriction sites, alterglycosylation patterns, or change codon preference, etc.).

Variants of RTP1 include but are not limited to RTP1-A, RTP1-B, RTP1-C,RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6).

TABLE 1 OR Modulator Genes Gene SEQ ID NO (Nucleic acid) SEQ ID NO(Polypeptide) Murine REEP1 1 21 Murine REEP2 2 22 Murine REEP3 3 23Murine REEP4 4 24 Murine REEP5 5 25 Murine REEP6 6 26 Human REEP1 7 27Human REEP2 8 28 Human REEP3 9 29 Human REEP4 10 30 Human REEP5 11 31Human REEP6 12 32 Murine RTP1 13 33 Murine RTP2 14 34 Murine RTP3 15 35Murine RTP4 16 36 Human RTP1 17 37 Human RTP2 18 38 Human RTP3 19 39Human RTP4 20 40III. REEP and RTP Polypeptides

In other embodiments, the present invention provides REEP and/or RTPpolynucleotide sequences that encode REEP and/or RTP polypeptidesequences (e.g., the polypeptides of SEQ ID NOs: 21-40, 41-50respectively). In preferred embodiments, the present invention providesa polypeptide encoded by a nucleic acid selected from the groupconsisting of SEQ ID NOs: 1, 7, 13, 14, 17 and 18 and variants thereofthat are at least 80% identical to SEQ ID NOs: 1, 7, 13, 14, 17 and 18.In further embodiments, the protein is at least 90% identical to SEQ IDNOs: 1, 7, 13, 14, 17 and 18. In even further embodiments, the proteinis at least 95% identical to SEQ ID NOs: 1, 7, 13, 14, 17 and 18. Otherembodiments of the present invention provide fragments, fusion proteinsor functional equivalents of REEP and/or RTP proteins (e.g., RTP1-A,RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3,RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3),RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera6). In some embodiments, the present invention provides mutants of REEPand/or RTP polypeptides. In still other embodiments of the presentinvention, nucleic acid sequences corresponding to REEP and/or RTPvariants, homologs, and mutants may be used to generate recombinant DNAmolecules that direct the expression of the REEP and/or RTP variants,homologs, and mutants in appropriate host cells. In some embodiments ofthe present invention, the polypeptide may be a naturally purifiedproduct, in other embodiments it may be a product of chemical syntheticprocedures, and in still other embodiments it may be produced byrecombinant techniques using a prokaryotic or eukaryotic host (e.g., bybacterial, yeast, higher plant, insect and mammalian cells in culture).In some embodiments, depending upon the host employed in a recombinantproduction procedure, the polypeptide of the present invention may beglycosylated or may be non-glycosylated. In other embodiments, thepolypeptides of the invention may also include an initial methionineamino acid residue.

In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than thepolynucleotide sequences of SEQ ID NOs: 21-50 that encode substantiallythe same or a functionally equivalent amino acid sequence, may be usedto clone and express REEP and/or RTP proteins. In general, suchpolynucleotide sequences hybridize to one of SEQ ID NOs: 21-50 underconditions of high to medium stringency as described above. As will beunderstood by those of skill in the art, it may be advantageous toproduce REEP and/or RTP-encoding nucleotide sequences possessingnon-naturally occurring codons. Therefore, in some preferredembodiments, codons preferred by a particular prokaryotic or eukaryotichost (Murray et al., Nucl. Acids Res., 17 [1989]) are selected, forexample, to increase the rate of REEP and/or RTP expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

In preferred embodiments, REEP1, RTP1 and RTP2 polypeptides promoteodorant receptor cell surface localization and odorant receptorfunctional expression.

1. Vectors for Production of REEP and RTP

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NOs: 1-20). In some embodiments ofthe present invention, the constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In still otherembodiments, the heterologous structural sequence (e.g., SEQ ID NOs:1-20 is assembled in appropriate phase with translation initiation andtermination sequences. In preferred embodiments of the presentinvention, the appropriate DNA sequence is inserted into the vectorusing any of a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1,pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3)Baculovirus—pPbac and pMbac (Stratagene). Any other plasmid or vectormay be used as long as they are replicable and viable in the host. Insome preferred embodiments of the present invention, mammalianexpression vectors comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation sites, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences. Inother embodiments, DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda P_(L) andP_(R), T3 and T7 promoters, and the cytomegalovirus (CMV) immediateearly, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein-I promoters and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.In other embodiments of the present invention, recombinant expressionvectors include origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or tetracycline orampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin bp 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

2. Host Cells for Production of REEP and RTP Polypeptides

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127,3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al., Basic Methodsin Molecular Biology, [1986]). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., [1989].

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

In preferred embodiments, the present invention provides a cell line(e.g., heterologous 293T cell line) comprising expression of an odorantreceptor (e.g., human odorant receptor, murine odorant receptor,synthetic odorant receptor) localized to the cell surface, REEP1, RTP1,RTP2, and G_(αolf). In some embodiments, the odorant receptor is taggedwith a reporting agent (e.g., glutathione-S-transferase (GST), c-myc,6-histidine (6X-His), green fluorescent protein (GFP), maltose bindingprotein (MBP), influenza A virus haemagglutinin (HA), b-galactosidase,and GAL4). The cell line described in this embodiment is not limited toparticular odorant receptors. In some embodiments, the odorant receptorsexpressed in the cell line include, but are not limited to, S6/79, S18,S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR32-11. In preferredembodiments, cell lines expressing odorant receptors are used in theclassification of an odorant receptor's functional expression (e.g.,ligand specificity). In even further embodiments, cell lines expressingodorant receptors are used in the classification of an animal'solfactory sensation.

3. Purification Of REEP and RTP Polypeptides

The present invention also provides methods for recovering and purifyingREEP and/or RTP polypeptides from recombinant cell cultures including,but not limited to, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. In other embodiments of the present invention,protein-refolding steps can be used as necessary, in completingconfiguration of the mature protein. In still other embodiments of thepresent invention, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The present invention further provides polynucleotides having a codingsequence of a REEP and/or RTP gene (e.g., SEQ ID NOs: 1-20) fused inframe to a marker sequence that allows for purification of thepolypeptide of the present invention. A non-limiting example of a markersequence is a hexahistidine tag which may be supplied by a vector,preferably a pQE-9 vector, which provides for purification of thepolypeptide fused to the marker in the case of a bacterial host, or, forexample, the marker sequence may be a hemagglutinin (HA) tag when amammalian host (e.g., COS-7 cells) is used. The HA tag corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,Cell, 37:767 [1984]).

4. Truncation Mutants of REEP and RTP Polypeptides

In addition, the present invention provides fragments of REEP and/or RTPpolypeptides (i.e., truncation mutants). In some embodiments of thepresent invention, when expression of a portion of the REEP and/or RTPprotein is desired, it may be necessary to add a start codon (ATG) tothe oligonucleotide fragment containing the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al., J. Bacteriol., 169:751 [1987]) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al., Proc. Natl. Acad. Sci. USA 84:2718[1990]). Therefore, removal of an N-terminal methionine, if desired, canbe achieved either in vivo by expressing such recombinant polypeptidesin a host which produces MAP (e.g., E. coli or CM89 or S. cerivisiae),or in vitro by use of purified MAP.

5. Fusion Proteins Containing REEP and RTP

The present invention also provides fusion proteins incorporating all orpart of the REEP and/or RTP polypeptides of the present invention.Accordingly, in some embodiments of the present invention, the codingsequences for the polypeptide can be incorporated as a part of a fusiongene including a nucleotide sequence encoding a different polypeptide.It is contemplated that this type of expression system will find useunder conditions where it is desirable to produce an immunogenicfragment of a REEP and/or RTP protein. In some embodiments of thepresent invention, the VP6 capsid protein of rotavirus is used as animmunologic carrier protein for portions of a REEP and/or RTPpolypeptide, either in the monomeric form or in the form of a viralparticle. In other embodiments of the present invention, the nucleicacid sequences corresponding to the portion of a REEP and/or RTPpolypeptide against which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising a portionof REEP and/or RTP as part of the virion. It has been demonstrated withthe use of immunogenic fusion proteins utilizing the hepatitis B surfaceantigen fusion proteins that recombinant hepatitis B virions can beutilized in this role as well. Similarly, in other embodiments of thepresent invention, chimeric constructs coding for fusion proteinscontaining a portion of a REEP and/or RTP polypeptide and the polioviruscapsid protein are created to enhance immunogenicity of the set ofpolypeptide antigens (See e.g., EP Publication No. 025949; and Evans etal., Nature 339:385 [1989]; Huang et al., J. Virol., 62:3855 [1988]; andSchlienger et al., J. Virol., 66:2 [1992]).

In still other embodiments of the present invention, the multipleantigen peptide system for peptide-based immunization can be utilized.In this system, a desired portion of REEP and/or RTP is obtaineddirectly from organo-chemical synthesis of the peptide onto anoligomeric branching lysine core (see e.g., Posnett et al., J. Biol.Chem., 263:1719 [1988]; and Nardelli et al., J. Immunol., 148:914[1992]). In other embodiments of the present invention, antigenicdeterminants of the REEP and/or RTP proteins can also be expressed andpresented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, such as a REEP and/or RTP protein of the presentinvention. Accordingly, in some embodiments of the present invention,REEP and/or RTP polypeptides can be generated asglutathione-S-transferase (i.e., GST fusion proteins). It iscontemplated that such GST fusion proteins will enable easy purificationof REEP and/or RTP polypeptides, such as by the use ofglutathione-derivatized matrices (See e.g., Ausabel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY [1991]).In another embodiment of the present invention, a fusion gene coding fora purification leader sequence, such as a poly-(H is)/enterokinasecleavage site sequence at the N-terminus of the desired portion of aREEP and/or RTP polypeptide, can allow purification of the expressedREEP and/or RTP fusion protein by affinity chromatography using a Ni²⁺metal resin. In still another embodiment of the present invention, thepurification leader sequence can then be subsequently removed bytreatment with enterokinase (See e.g., Hochuli et al., J. Chromatogr.,411:177 [1987]; and Janknecht et al., Proc. Natl. Acad. Sci. USA88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment ofthe present invention, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, in other embodiments of the present invention, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (See e.g., Current Protocols in Molecular Biology, supra).

6. Variants of REEP and RTP

Still other embodiments of the present invention provide mutant orvariant forms of REEP and/or RTP polypeptides (i.e., muteins). It ispossible to modify the structure of a peptide having an activity of aREEP and/or RTP polypeptide of the present invention for such purposesas enhancing therapeutic or prophylactic efficacy, disabling theprotein, or stability (e.g., ex vivo shelf life, and/or resistance toproteolytic degradation in vivo). Such modified peptides are consideredfunctional equivalents of peptides having an activity of the subjectREEP and/or RTP proteins as defined herein. A modified peptide can beproduced in which the amino acid sequence has been altered, such as byamino acid substitution, deletion, or addition.

Variant forms of RTP1 include but are not limited to RTP1-A, RTP1-B,RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A(Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A(Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6).

Moreover, as described above, variant forms (e.g., mutants orpolymorphic sequences) of the subject REEP and/or RTP proteins are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail. For example, as described above, thepresent invention encompasses mutant and variant proteins that containconservative or non-conservative amino acid substitutions.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the present REEP and/or RTP proteins, as wellas truncation mutants, and is especially useful for identifyingpotential variant sequences (i.e., mutants or polymorphic sequences)that are involved in neurological disorders (e.g., olfactory disorders)or resistance to neurological disorders. The purpose of screening suchcombinatorial libraries is to generate, for example, novel REEP and/orRTP variants that can act as either agonists or antagonists, oralternatively, possess novel activities all together.

Therefore, in some embodiments of the present invention, REEP and/or RTPvariants are engineered by the present method to provide altered (e.g.,increased or decreased) biological activity. In other embodiments of thepresent invention, combinatorially-derived variants are generated whichhave a selective potency relative to a naturally occurring REEP and/orRTP. Such proteins, when expressed from recombinant DNA constructs, canbe used in gene therapy protocols.

Still other embodiments of the present invention provide REEP and/or RTPvariants that have intracellular half-lives dramatically different thanthe corresponding wild-type protein. For example, the altered proteincan be rendered either more stable or less stable to proteolyticdegradation or other cellular process that result in destruction of, orotherwise inactivate REEP and/or RTP polypeptides. Such variants, andthe genes which encode them, can be utilized to alter the location ofREEP and/or RTP expression by modulating the half-life of the protein.For instance, a short half-life can give rise to more transient REEPand/or RTP biological effects and, when part of an inducible expressionsystem, can allow tighter control of REEP and/or RTP levels within thecell. As above, such proteins, and particularly their recombinantnucleic acid constructs, can be used in gene therapy protocols.

In still other embodiments of the present invention, REEP and/or RTPvariants are generated by the combinatorial approach to act asantagonists, in that they are able to interfere with the ability of thecorresponding wild-type protein to regulate cell function.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of REEPand/or RTP homologs, variants or other related proteins are aligned,preferably to promote the highest homology possible. Such a populationof variants can include, for example, REEP and/or RTP homologs from oneor more species, or REEP and/or RTP variants from the same species butwhich differ due to mutation or polymorphisms. Amino acids that appearat each position of the aligned sequences are selected to create adegenerate set of combinatorial sequences.

In a preferred embodiment of the present invention, the combinatorialREEP and/or RTP library is produced by way of a degenerate library ofgenes encoding a library of polypeptides which each include at least aportion of potential REEP and/or RTP protein sequences. For example, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential REEP and/or RTPsequences are expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (e.g., for phage display) containingthe set of REEP and/or RTP sequences therein.

There are many ways by which the library of potential REEP and/or RTPhomologs and variants can be generated from a degenerate oligonucleotidesequence. In some embodiments, chemical synthesis of a degenerate genesequence is carried out in an automatic DNA synthesizer, and thesynthetic genes are ligated into an appropriate gene for expression. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential REEP and/or RTPsequences. The synthesis of degenerate oligonucleotides is well known inthe art (See e.g., Narang, Tetrahedron Lett., 39:39 [1983]; Itakura etal., Recombinant DNA, in Walton (ed.), Proceedings of the 3rd ClevelandSymposium on Macromolecules, Elsevier, Amsterdam, pp 273-289 [1981];Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura et al.,Science 198:1056 [1984]; Ike et al., Nucl. Acid Res., 11:477 [1983]).Such techniques have been employed in the directed evolution of otherproteins (See e.g., Scott et al., Science 249:386 [1980]; Roberts etal., Proc. Natl. Acad. Sci. USA 89:2429 [1992]; Devlin et al., Science249: 404 [1990]; Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378[1990]; each of which is herein incorporated by reference; as well asU.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815; each of which isincorporated herein by reference).

It is contemplated that the REEP and/or RTP nucleic acids of the presentinvention (e.g., SEQ ID NOs: 1-20, and fragments and variants thereof)can be utilized as starting nucleic acids for directed evolution. Thesetechniques can be utilized to develop REEP and/or RTP variants havingdesirable properties such as increased or decreased biological activity.

In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive enzyme. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold, Nat. Biotech., 14, 458 [1996]; Leung et al., Technique, 1:11[1989]; Eckert and Kunkel, PCR Methods Appl., 1:17-24 [1991]; Caldwelland Joyce, PCR Methods Appl., 2:28 [1992]; and Zhao and Arnold, Nuc.Acids. Res., 25:1307 [1997]). After mutagenesis, the resulting clonesare selected for desirable activity (e.g., screened for REEP and/or RTPactivity). Successive rounds of mutagenesis and selection are oftennecessary to develop enzymes with desirable properties. It should benoted that only the useful mutations are carried over to the next roundof mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith, Nature, 370:324 [1994]; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer, Nature, 370:398[1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91:10747 [1994]; Crameri etal., Nat. Biotech., 14:315 [1996]; Zhang et al., Proc. Natl. Acad. Sci.USA, 94:4504 [1997]; and Crameri et al., Nat. Biotech., 15:436 [1997]).Variants produced by directed evolution can be screened for REEP and/orRTP activity by the methods described herein.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis orrecombination of REEP and/or RTP homologs or variants. The most widelyused techniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected.

7. Chemical Synthesis of REEP and/or RTP Polypeptides

In an alternate embodiment of the invention, the coding sequence of REEPand/or RTP is synthesized, whole or in part, using chemical methods wellknown in the art (See e.g., Caruthers et al., Nucl. Acids Res. Symp.Ser., 7:215 [1980]; Crea and Horn, Nucl. Acids Res., 9:2331 [1980];Matteucci and Caruthers, Tetrahedron Lett., 21:719 [1980]; and Chow andKempe, Nucl. Acids Res., 9:2807 [1981]). In other embodiments of thepresent invention, the protein itself is produced using chemical methodsto synthesize either an entire REEP and/or RTP amino acid sequence or aportion thereof. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (See e.g., Creighton, ProteinsStructures And Molecular Principles, W H Freeman and Co, New York N.Y.[1983]). In other embodiments of the present invention, the compositionof the synthetic peptides is confirmed by amino acid analysis orsequencing (See e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al., Science 269:202 [1995]) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequence of a REEPand/or RTP polypeptide, or any part thereof, may be altered duringdirect synthesis and/or combined using chemical methods with othersequences to produce a variant polypeptide.

IV. Detection of REEP and RTP Alleles

In some embodiments, the present invention provides methods of detectingthe presence of wild type or variant (e.g., mutant or polymorphic) REEPand/or RTP nucleic acids or polypeptides. The detection of mutant REEPand/or RTP polypeptides finds use in the diagnosis of disease (e.g.,olfactory disorder).

A. Detection of Variant REEP and/or RTP Alleles

In some embodiments, the present invention provides alleles of REEPand/or RTP that increase a patient's susceptibility to olfactorydisorders (e.g., upper respiratory infections, tumors of the anteriorcranial fossa, Kallmann syndrome, Foster Kennedy syndrome, Parkinson'sdisease, Alzheimer's disease, and Huntington chorea). Any mutation thatresults in an altered phenotype (e.g., diminished olfactory sensingability) is within the scope of the present invention.

Accordingly, the present invention provides methods for determiningwhether a patient has an increased susceptibility to olfactory disorders(e.g., upper respiratory infections, tumors of the anterior cranialfossa, and Kallmann syndrome, Foster Kennedy syndrome, Parkinson'sdisease, Alzheimer's disease, Huntington chorea) by determining,directly or indirectly, whether the individual has a variant REEP and/orRTP allele. In other embodiments, the present invention provides methodsfor providing a prognosis of increased risk for olfactory disorder to anindividual based on the presence or absence of one or more variant REEPand/or RTP alleles.

A number of methods are available for analysis of variant (e.g., mutantor polymorphic) nucleic acid or polypeptide sequences. Assays fordetection variants (e.g., polymorphisms or mutations) via nucleic acidanalysis fall into several categories including, but not limited to,direct sequencing assays, fragment polymorphism assays, hybridizationassays, and computer based data analysis. Protocols and commerciallyavailable kits or services for performing multiple variations of theseassays are available. In some embodiments, assays are performed incombination or in hybrid (e.g., different reagents or technologies fromseveral assays are combined to yield one assay). The following exemplaryassays are useful in the present invention: directs sequencing assays,PCR assays, mutational analysis by dHPLC (e.g., available fromTransgenomic, Omaha, Nebr. or Varian, Palo Alto, Calif.), fragmentlength polymorphism assays (e.g., RFLP or CFLP (See e.g. U.S. patentsU.S. Pat. Nos. 5,843,654; 5,843,669; 5,719,208; and 5,888,780; each ofwhich is herein incorporated by reference)), hybridization assays (e.g.,direct detection of hybridization, detection of hybridization using DNAchip assays (See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; 5,858,659;6,017,696; 6,068,818; 6,051,380; 6,001,311; 5,985,551; 5,474,796; PCTPublications WO 99/67641 and WO 00/39587, each of which is hereinincorporated by reference), enzymatic detection of hybridization (Seee.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557;5,994,069; 5,962,233; 5,538,848; 5,952,174 and 5,919,626, each of whichis herein incorporated by reference)), polymorphisms detected directlyor indirectly (e.g., detecting sequences (other polymorphisms) that arein linkage disequilibrium with the polymorphism to be identified; forexample, other sequences in the SPG-6 locus may be used; this method isdescribed in U.S. Pat. No. 5,612,179 (herein incorporated by reference))and mass spectrometry assays.

In addition, assays for the detection of variant REEP and/or RTPproteins find use in the present invention (e.g., cell free translationmethods, See e.g., U.S. Pat. No. 6,303,337, herein incorporated byreference) and antibody binding assays. The generation of antibodiesthat specifically recognize mutant versus wild type proteins arediscussed below.

B. Kits for Analyzing Risk of Olfactory Disorders

The present invention also provides kits for determining whether anindividual contains a wild-type or variant (e.g., mutant or polymorphic)allele or polypeptide of REEP and/or RTP. In some embodiments, the kitsare useful determining whether the subject is at risk of developing anolfactory disorder (e.g., upper respiratory infections, tumors of theanterior cranial fossa, and Kallmann syndrome, Foster Kennedy syndrome,Parkinson's disease, Alzheimer's disease, Huntington chorea). Thediagnostic kits are produced in a variety of ways. In some embodiments,the kits contain at least one reagent for specifically detecting amutant REEP and/or RTP allele or protein. In preferred embodiments, thereagent is a nucleic acid that hybridizes to nucleic acids containingthe mutation and that does not bind to nucleic acids that do not containthe mutation. In other embodiments, the reagents are primers foramplifying the region of DNA containing the mutation. In still otherembodiments, the reagents are antibodies that preferentially bind eitherthe wild-type or mutant REEP and/or RTP proteins.

In some embodiments, the kit contains instructions for determiningwhether the subject is at risk for an olfactory disorder (e.g., upperrespiratory infections, tumors of the anterior cranial fossa, andKallmann syndrome, Foster Kennedy syndrome, Parkinson's disease,Alzheimer's disease, Huntington chorea). In preferred embodiments, theinstructions specify that risk for developing an olfactory disorder isdetermined by detecting the presence or absence of a mutant REEP and/orRTP allele in the subject, wherein subjects having an mutant allele areat greater risk for developing an olfactory disorder.

The presence or absence of a disease-associated mutation in a REEPand/or RTP gene can be used to make therapeutic or other medicaldecisions. For example, couples with a family history of odorantreceptor related diseases may choose to conceive a child via in vitrofertilization and pre-implantation genetic screening. In this case,fertilized embryos are screened for mutant (e.g., disease associated)alleles of a REEP and/or RTP gene and only embryos with wild typealleles are implanted in the uterus.

In other embodiments, in utero screening is performed-on a developingfetus (e.g., amniocentesis or chorionic villi screening). In still otherembodiments, genetic screening of newborn babies or very young childrenis performed. The early detection of a REEP and/or RTP allele known tobe associated with an olfactory disorder allows for early intervention(e.g., genetic or pharmaceutical therapies).

In some embodiments, the kits include ancillary reagents such asbuffering agents, nucleic acid stabilizing reagents, protein stabilizingreagents, and signal producing systems (e.g., florescence generatingsystems as Fret systems). The test kit may be packaged in any suitablemanner, typically with the elements in a single container or variouscontainers as necessary along with a sheet of instructions for carryingout the test. In some embodiments, the kits also preferably include apositive control sample.

C. Bioinformatics

In some embodiments, the present invention provides methods ofdetermining an individual's risk of developing an olfactory disorder(e.g., upper respiratory infections, tumors of the anterior cranialfossa, and Kallmann syndrome, Foster Kennedy syndrome, Parkinson'sdisease, Alzheimer's disease, Huntington chorea) based on the presenceof one or more variant alleles of a REEP and/or RTP gene. In someembodiments, the analysis of variant data is processed by a computerusing information stored on a computer (e.g., in a database). Forexample, in some embodiments, the present invention provides abioinformatics research system comprising a plurality of computersrunning a multi-platform object oriented programming language (See e.g.,U.S. Pat. No. 6,125,383; herein incorporated by reference). In someembodiments, one of the computers stores genetics data (e.g., the riskof contacting an REEP and/or RTP related olfactory disorder associatedwith a given polymorphism, as well as the sequences). In someembodiments, one of the computers stores application programs (e.g., foranalyzing the results of detection assays). Results are then deliveredto the user (e.g., via one of the computers or via the internet).

For example, in some embodiments, a computer-based analysis program isused to translate the raw data generated by the detection assay (e.g.,the presence, absence, or amount of a given REEP and/or RTP allele orpolypeptide) into data of predictive value for a clinician. Theclinician can access the predictive data using any suitable means. Thus,in some preferred embodiments, the present invention provides thefurther benefit that the clinician, who is not likely to be trained ingenetics or molecular biology, need not understand the raw data. Thedata is presented directly to the clinician in its most useful form. Theclinician is then able to immediately utilize the information in orderto optimize the care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information providers, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., presence of wild type ormutant REEP and/or RTP genes or polypeptides), specific for thediagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw data, the prepared format may represent a diagnosis orrisk assessment (e.g., likelihood of developing an REEP and/or RTPrelated olfactory disorder) for the subject, along with recommendationsfor particular treatment options. The data may be displayed to theclinician by any suitable method. For example, in some embodiments, theprofiling service generates a report that can be printed for theclinician (e.g., at the point of care) or displayed to the clinician ona computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the association of a given REEP and/or RTP allele witholfactory disorders.

V. Generation of REEP and RTP Antibodies

The present invention provides isolated antibodies or antibody fragments(e.g., FAB fragments). Antibodies can be generated to allow for thedetection of REEP and/or RTP proteins (e.g., wild type or mutant) of thepresent invention. The antibodies may be prepared using variousimmunogens. In one embodiment, the immunogen is a human REEP and/or RTPpeptide to generate antibodies that recognize human REEP and/or RTP.Such antibodies include, but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, Fab expression libraries, orrecombinant (e.g., chimeric, humanized, etc.) antibodies, as long as itcan recognize the protein. Antibodies can be produced by using a proteinof the present invention as the antigen according to a conventionalantibody or antiserum preparation process.

Various procedures known in the art may be used for the production ofpolyclonal antibodies directed against a REEP and/or RTP polypeptide.For the production of antibody, various host animals can be immunized byinjection with the peptide corresponding to the REEP and/or RTP epitopeincluding but not limited to rabbits, mice, rats, sheep, goats, etc. Ina preferred embodiment, the peptide is conjugated to an immunogeniccarrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyholelimpet hemocyanin (KLH)). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).

For preparation of monoclonal antibodies directed toward REEP and/orRTP, it is contemplated that any technique that provides for theproduction of antibody molecules by continuous cell lines in culturewill find use with the present invention (See e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). These include but are not limited to thehybridoma technique originally developed by Köhler and Milstein (Köhlerand Milstein, Nature 256:495-497 [1975]), as well as the triomatechnique, the human B-cell hybridoma technique (See e.g., Kozbor etal., Immunol. Tod., 4:72 [1983]), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 [1985]).

In an additional embodiment of the invention, monoclonal antibodies areproduced in germ-free animals utilizing technology such as thatdescribed in PCT/US90/02545). Furthermore, it is contemplated that humanantibodies will be generated by human hybridomas (Cote et al., Proc.Natl. Acad. Sci. USA 80:2026-2030 [1983]) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77-96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing REEP and/or RTPspecific single chain antibodies. An additional embodiment of theinvention utilizes the techniques described for the construction of Fabexpression libraries (Huse et al., Science 246:1275-1281 [1989]) toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for a REEP and/or RTP polypeptide.

In other embodiments, the present invention contemplated recombinantantibodies or fragments thereof to the proteins of the presentinvention. Recombinant antibodies include, but are not limited to,humanized and chimeric antibodies. Methods for generating recombinantantibodies are known in the art (See e.g., U.S. Pat. Nos. 6,180,370 and6,277,969 and “Monoclonal Antibodies” H. Zola, BIOS ScientificPublishers Limited 2000. Springer-Verlay New York, Inc., New York; eachof which is herein incorporated by reference).

It is contemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that containthe idiotype (antigen binding region) of the antibody molecule. Forexample, such fragments include but are not limited to: F(ab′)2 fragmentthat can be produced by pepsin digestion of the antibody molecule; Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and Fab fragments that can be generated by treatingthe antibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening forthe desired antibody will be accomplished by techniques known in the art(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situ immunoassays(e.g., using colloidal gold, enzyme or radioisotope labels), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.)

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of REEP and/or RTP (e.g., forWestern blotting), measuring levels thereof in appropriate biologicalsamples, etc. The antibodies can be used to detect a REEP and/or RTP ina biological sample from an individual. The biological sample can be abiological fluid, such as, but not limited to, blood, serum, plasma,interstitial fluid, urine, cerebrospinal fluid, and the like, containingcells.

The biological samples can then be tested directly for the presence of ahuman REEP and/or RTP using an appropriate strategy (e.g., ELISA orradioimmunoassay) and format (e.g., microwells, dipstick (e.g., asdescribed in International Patent Publication WO 93/03367), etc.Alternatively, proteins in the sample can be size separated (e.g., bypolyacrylamide gel electrophoresis (PAGE), in the presence or not ofsodium dodecyl sulfate (SDS), and the presence of REEP and/or RTPdetected by immunoblotting (Western blotting). Immunoblotting techniquesare generally more effective with antibodies generated against a peptidecorresponding to an epitope of a protein, and hence, are particularlysuited to the present invention.

Another method uses antibodies as agents to alter signal transduction.Specific antibodies that bind to the binding domains of REEP and/or RTPor other proteins involved in intracellular signaling can be used toinhibit the interaction between the various proteins and theirinteraction with other ligands. Antibodies that bind to the complex canalso be used therapeutically to inhibit interactions of the proteincomplex in the signal transduction pathways leading to the variousphysiological and cellular effects of REEP and/or RTP. Such antibodiescan also be used diagnostically to measure abnormal expression of REEP1and/or RTP, or the aberrant formation of protein complexes, which may beindicative of a disease state.

VI. Gene Therapy Using REEP and RTP

The present invention also provides methods and compositions suitablefor gene therapy to alter REEP and/or RTP expression, production, orfunction for research, generation of transgenic animals, and/ortherapeutic applications. As described above, the present inventionprovides human REEP and/or RTP genes and provides methods of obtainingREEP and/or RTP genes from other species. Thus, the methods describedbelow are generally applicable across many species. In some embodiments,it is contemplated that the gene therapy is performed by providing asubject with a wild-type allele of a REEP and/or RTP gene (i.e., anallele that does not contain a REEP and/or RTP disease allele (e.g.,free of disease causing polymorphisms or mutations)). Subjects in needof such therapy are identified by the methods described above. In someembodiments, transient or stable therapeutic nucleic acids are used(e.g., antisense oligonucleotides, siRNAs) to reduce or preventexpression of mutant proteins. In other embodiments, genes are deletedto reduce or block desired olfactory senses.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (See e.g.,Miller and Rosman, BioTech., 7:980-990 [1992]). Preferably, the viralvectors are replication defective, that is, they are unable to replicateautonomously in the target cell. In general, the genome of thereplication defective viral vectors that are used within the scope ofthe present invention lack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (i.e., on the isolated DNA) or insitu, using the techniques of genetic manipulation or by treatment withmutagenic agents.

Preferably, the replication defective virus retains the sequences of itsgenome that are necessary for encapsidating the viral particles. DNAviral vectors include an attenuated or defective DNA viruses, including,but not limited to, herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, that entirely or almost entirely lack viralgenes, are preferred, as defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Mol. Cell. Neurosci., 2:320-330 [1991]), defective herpes virusvector lacking a glycoprotein L gene (See e.g., Patent Publication RD371005 A), or other defective herpes virus vectors (See e.g., WO94/21807; and WO 92/05263); an attenuated adenovirus vector, such as thevector described by Stratford-Perricaudet et al. (J. Clin. Invest.,90:626-630 [1992]; See also, La Salle et al., Science 259:988-990[1993]); and a defective adeno-associated virus vector (Samulski et al.,J. Virol., 61:3096-3101 [1987]; Samulski et al., J. Virol., 63:3822-3828[1989]; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 [1988]).

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector (e.g.,adenovirus vector), to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-gamma (IFN-γ), or anti-CD4 antibody,can be administered to block humoral or cellular immune responses to theviral vectors. In addition, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector.Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to type2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animalorigin (See e.g., WO 94/26914). Those adenoviruses of animal origin thatcan be used within the scope of the present invention includeadenoviruses of canine, bovine, murine (e.g., Mav1, Beard et al.,Virol., 75-81 [1990]), ovine, porcine, avian, and simian (e.g., SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61strain (ATCC VR-800)).

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3Afragment). Other regions may also be modified, in particular the E3region (e.g., WO 95/02697), the E2 region (e.g., WO 94/28938), the E4region (e.g., WO 94/28152, WO 94/12649 and WO 95/02697), or in any ofthe late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed inEP 185,573, the contents of which are incorporated herein by reference.In another preferred embodiment, the adenoviral vector has a deletion inthe E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenovirusesare disclosed in WO 95/02697 and WO 96/22378. In still another preferredembodiment, the adenoviral vector has a deletion in the E1 region intowhich the E4 region and the nucleic acid sequence are inserted.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (See e.g., Levrero et al., Gene 101:195 [1991]; EP 185 573;and Graham, EMBO J., 3:2917 [1984]). In particular, they can be preparedby homologous recombination between an adenovirus and a plasmid thatcarries, inter alia, the DNA sequence of interest. The homologousrecombination is accomplished following co-transfection of theadenovirus and plasmid into an appropriate cell line. The cell line thatis employed should preferably (i) be transformable by the elements to beused, and (ii) contain the sequences that are able to complement thepart of the genome of the replication defective adenovirus, preferablyin integrated form in order to avoid the risks of recombination.Examples of cell lines that may be used are the human embryonic kidneycell line 293 (Graham et al., J. Gen. Virol., 36:59 [1977]), whichcontains the left-hand portion of the genome of an Ad5 adenovirus (12%)integrated into its genome, and cell lines that are able to complementthe E1 and E4 functions, as described in applications WO 94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques that are well known to one ofordinary skill in the art.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize that can integrate, in a stable and site-specific manner, into thegenome of the cells that they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. It encompasses approximately 4700 bases and contains aninverted terminal repeat (ITR) region of approximately 145 bases at eachend, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions that carrythe encapsidation functions: the left-hand part of the genome, thatcontains the rep gene involved in viral replication and expression ofthe viral genes; and the right-hand part of the genome, that containsthe cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S.Pat. No. 4,797,368; U.S. Pat. No. 5,139,941; and EP 488 528, all ofwhich are herein incorporated by reference). These publications describevarious AAV-derived constructs in which the rep and/or cap genes aredeleted and replaced by a gene of interest, and the use of theseconstructs for transferring the gene of interest in vitro (into culturedcells) or in vivo (directly into an organism). The replication defectiverecombinant AAVs according to the invention can be prepared byco-transfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (ITR) regions, anda plasmid carrying the AAV encapsidation genes (rep and cap genes), intoa cell line that is infected with a human helper virus (for example anadenovirus). The AAV recombinants that are produced are then purified bystandard techniques.

In another embodiment, the gene can be introduced in a retroviral vector(e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289and 5,124,263; all of which are herein incorporated by reference; Mannet al., Cell 33:153 [1983]; Markowitz et al., J. Virol., 62:1120 [1988];PCT/US95/14575; EP 453242; EP178220; Bernstein et al. Genet. Eng., 7:235[1985]; McCormick, BioTechnol., 3:689 [1985]; WO 95/07358; and Kuo etal., Blood 82:845 [1993]). The retroviruses are integrating viruses thatinfect dividing cells. The retrovirus genome includes two LTRs, anencapsidation sequence and three coding regions (gag, pol and env). Inrecombinant retroviral vectors, the gag, pol and env genes are generallydeleted, in whole or in part, and replaced with a heterologous nucleicacid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as, HIV, MoMuLV (“murine Moloneyleukemia virus” MSV (“murine Moloney sarcoma virus”), HaSV (“Harveysarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcomavirus”) and Friend virus. Defective retroviral vectors are alsodisclosed in WO 95/02697.

In general, in order to construct recombinant retroviruses containing anucleic acid sequence, a plasmid is constructed that contains the LTRs,the encapsidation sequence and the coding sequence. This construct isused to transfect a packaging cell line, which cell line is able tosupply in trans the retroviral functions that are deficient in theplasmid. In general, the packaging cell lines are thus able to expressthe gag, pol and env genes. Such packaging cell lines have beendescribed in the prior art, in particular the cell line PA317 (U.S. Pat.No. 4,861,719, herein incorporated by reference), the PsiCRIP cell line(See, WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences that may include a part of the gaggene (Bender et al., J. Virol., 61:1639 [1987]). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al., Proc. Natl. Acad. Sci. USA 84:7413-7417 [1987]; See also,Mackey, et al., Proc. Natl. Acad. Sci. USA 85:8027-8031 [1988]; Ulmer etal., Science 259:1745-1748 [1993]). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, Science 337:387-388 [1989]). Particularly useful lipidcompounds and compositions for transfer of nucleic acids are describedin WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, hereinincorporated by reference.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Methods for formulating and administering naked DNA tomammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and5,589,466, both of which are herein incorporated by reference.

DNA vectors for gene therapy can be introduced into the desired hostcells by methods known in the art, including but not limited totransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (See e.g., Wu et al., J. Biol.Chem., 267:963 [1992]; Wu and Wu, J. Biol. Chem., 263:14621 [1988]; andWilliams et al., Proc. Natl. Acad. Sci. USA 88:2726 [1991]).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther., 3:147 [1992]; and Wu and Wu, J. Biol. Chem.,262:4429 [1987]).

VII. Transgenic Animals Expressing Exogenous REEP and RTP Genes andHomologs, Mutants, and Variants Thereof

The present invention contemplates the generation of transgenic animalscomprising an exogenous REEP and/or RTP gene or homologs, mutants, orvariants thereof. In preferred embodiments, the transgenic animaldisplays an altered phenotype as compared to wild-type animals. In someembodiments, the altered phenotype is the overexpression of mRNA for aREEP and/or RTP gene as compared to wild-type levels of REEP and/or RTPexpression. In other embodiments, the altered phenotype is the decreasedexpression of mRNA for an endogenous REEP and/or RTP gene as compared towild-type levels of endogenous REEP and/or RTP expression. In somepreferred embodiments, the transgenic animals comprise mutant alleles ofREEP and/or RTP. Methods for analyzing the presence or absence of suchphenotypes include Northern blotting, mRNA protection assays, andRT-PCR. In other embodiments, the transgenic mice have a knock outmutation of a REEP and/or RTP gene. In preferred embodiments, thetransgenic animals display an altered susceptibility to olfactorydisorders (e.g., upper respiratory infections, tumors of the anteriorcranial fossa, and Kallmann syndrome, Foster Kennedy syndrome,Parkinson's disease, Alzheimer's disease, Huntington chorea).

Such animals find use in research applications (e.g., identifyingsignaling pathways that a REEP and/or RTP protein is involved in), aswell as drug screening applications (e.g., to screen for drugs thatprevent or treat olfactory disorders). For example, in some embodiments,test compounds (e.g., a drug that is suspected of being useful to treatan olfactory disorder) are administered to the transgenic animals andcontrol animals with a wild type REEP and/or RTP allele and the effectsevaluated. The effects of the test and control compounds on diseasesymptoms are then assessed.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter, which allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al., EMBO J., 6:383 [1987]). Alternatively, infectioncan be performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al., Nature 298:623 [1982]).Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells that form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal., supra [1982]). Additional means of using retroviruses or retroviralvectors to create transgenic animals known to the art involves themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (PCT International Application WO 90/08832 [1990], andHaskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or create deletion mutants (e.g., mutants inwhich a particular domain of REEP and/or RTP is deleted). Methods forhomologous recombination are described in U.S. Pat. No. 5,614,396,incorporated herein by reference.

VIII. Compound Screening Using REEP and RTP

In some embodiments, the isolated nucleic acid and polypeptides of REEPand/or RTP genes of the present invention (e.g., SEQ ID NOS: 1-50) andrelated proteins and nucleic acids are used in drug screeningapplications for compounds that alter (e.g., enhance or inhibit) REEPand/or RTP activity and signaling. The present invention furtherprovides methods of identifying ligands and signaling pathways of theREEP and/or RTP proteins of the present invention.

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, based upon OR expression analysisexperiments conducted during the course of the present invention, it iscontemplated that REEP and/or RTP family proteins function in promotingodorant receptor cell surface localization and functional expression.

In some embodiments, the present invention provides methods of screeningcompounds for the ability to alter REEP and/or RTP activity mediated bynatural ligands (e.g., identified using the methods described above).Such compounds find use in the treatment of disease mediated by REEPand/or RTP (e.g., olfactory disorders), the alteration of olfactorysensory responses, and the like.

In some embodiments, the present invention provides methods of screeningcompounds for an ability to interact with mutant REEP and/or RTP nucleicacid and/or mutant REEP and/or RTP polypeptides, while simultaneouslynot interacting with wild type REEP and/or RTP nucleic acid (e.g., SEQID NOS: 1-20) and/or wild type REEP and/or RTP polypeptides (e.g., SEQID NOS:21-50). Such compounds find use in the treatment of olfactorydisorders facilitated by the presence of mutant forms of REEP and/or RTPnucleic acids and/or proteins.

In some embodiments, the activity of cell surface localized ORs in cellsexpressing exogenous REEP or RTP polypeptides is assessed in response tocompounds (e.g., candidate or ligands or inhibitors).

One technique uses REEP, RTP, or OR antibodies, generated as discussedabove. Such antibodies are capable of specifically binding to REEP, RTP,or OR peptides and compete with a test compound for binding to REEP,RTP, or OR peptides. Similar screens can be carried out with smallmolecule libraries, aptamers, etc.

The present invention contemplates the use of cell lines transfectedwith REEP and/or RTP genes and variants thereof for screening compoundsfor activity, and in particular to high throughput screening ofcompounds from combinatorial libraries (e.g., libraries containinggreater than 10⁴ compounds). The cell lines of the present invention canbe used in a variety of screening methods. In some embodiments, thecells can be used in second messenger assays that monitor signaltransduction following activation of cell-surface receptors. In otherembodiments, the cells can be used in reporter gene assays that monitorcellular responses at the transcription/translation level.

In second messenger assays, the host cells are preferably transfected asdescribed above with vectors encoding REEP and/or RTP or variants ormutants thereof. The host cells are then treated with a compound orplurality of compounds (e.g., from a combinatorial library) and assayedfor the presence or absence of a response. It is contemplated that atleast some of the compounds in the combinatorial library can serve asagonists, antagonists, activators, or inhibitors of the protein orproteins encoded by the vectors or of ORs localized at the cellmembrane. It is also contemplated that at least some of the compounds inthe combinatorial library can serve as agonists, antagonists,activators, or inhibitors of protein acting upstream or downstream ofthe protein encoded by the vector in a signal transduction pathway.

In some embodiments, the second messenger assays measure fluorescentsignals from reporter molecules that respond to intracellular changes(e.g., Ca²⁺ concentration, membrane potential, pH, IP₃, CAMP,arachidonic acid release) due to stimulation of membrane receptors andion channels (e.g., ligand gated ion channels; see Denyer et al., DrugDiscov. Today 3:323 [1998]; and Gonzales et al., Drug. Discov. Today4:431-39 [1999]). Examples of reporter molecules include, but are notlimited to, FRET (florescence resonance energy transfer) systems (e.g.,Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitiveindicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), andpH sensitive indicators (e.g., BCECF).

In general, the host cells are loaded with the indicator prior toexposure to the compound. Responses of the host cells to treatment withthe compounds can be detected by methods known in the art, including,but not limited to, fluorescence microscopy, confocal microscopy (e.g.,FCS systems), flow cytometry, microfluidic devices, FLIPR systems (See,e.g., Schroeder and Neagle, J. Biomol. Screening 1:75 [1996]), andplate-reading systems. In some preferred embodiments, the response(e.g., increase in fluorescent intensity) caused by compound of unknownactivity is compared to the response generated by a known agonist andexpressed as a percentage of the maximal response of the known agonist.The maximum response caused by a known agonist is defined as a 100%response. Likewise, the maximal response recorded after addition of anagonist to a sample containing a known or test antagonist is detectablylower than the 100% response.

The cells are also useful in reporter gene assays. Reporter gene assaysinvolve the use of host cells transfected with vectors encoding anucleic acid comprising transcriptional control elements of a targetgene (i.e., a gene that controls the biological expression and functionof a disease target) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

The ability of the test compound to modulate REEP and/or RTP binding toa compound, e.g., an odorant receptor, can also be evaluated. This canbe accomplished, for example, by coupling the compound, e.g., thesubstrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g., the substrate, to REEP and/or RTP can be determinedby detecting the labeled compound, e.g., substrate, in a complex.

Alternatively, REEP and/or RTP is coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulateREEP and/or RTP binding to a REEP and/or RTP substrate in a complex. Forexample, compounds (e.g., substrates) can be labeled with ¹²⁵I, ³⁵S ¹⁴Cor ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

The ability of a compound (e.g., an odorant receptor) to interact withREEP and/or RTP with or without the labeling of any of the interactantscan be evaluated. For example, a microphysiometer can be used to detectthe interaction of a compound with REEP and/or RTP without the labelingof either the compound or the REEP and/or RTP (McConnell et al. Science257:1906-1912 [1992]). As used herein, a “microphysiometer” (e.g.,Cytosensor) is an analytical instrument that measures the rate at whicha cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and a REEPand/or RTP polypeptide.

In yet another embodiment, a cell-free assay is provided in which REEPand/or RTP protein or biologically active portion thereof is contactedwith a test compound and the ability of the test compound to bind to theREEP and/or RTP protein or biologically active portion thereof isevaluated. Preferred biologically active portions of REEP and/or RTPproteins to be used in assays of the present invention include fragmentsthat participate in interactions with substrates or other proteins,e.g., fragments with high surface probability scores.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FRET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,968,103; each of which is herein incorporated by reference). Afluorophore label is selected such that a first donor molecule's emittedfluorescent energy will be absorbed by a fluorescent label on a second,‘acceptor’ molecule, which in turn is able to fluoresce due to theabsorbed energy.

Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in 1 5 theassay should be maximal. An FRET binding event can be convenientlymeasured through standard fluorometric detection means well known in theart (e.g., using a fluorimeter).

Modulators of REEP and/or RTP expression can also be identified. Forexample, a cell or cell free mixture is contacted with a candidatecompound and the expression of REEP and/or RTP mRNA or protein evaluatedrelative to the level of expression of the REEP and/or RTP mRNA orprotein in the absence of the candidate compound. When expression of theREEP and/or RTP mRNA or protein is greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of a REEP and/or RTP mRNA or proteinexpression. Alternatively, when expression of REEP and/or RTP mRNA orprotein is less (i.e., statistically significantly less) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as an inhibitor of REEP and/or RTP mRNA or proteinexpression. The level of REEP and/or RTP mRNA or protein expression canbe determined by methods described herein for detecting REEP and/or RTPmRNA or protein.

A modulating agent can be identified using a cell-based or a cell freeassay, and the ability of the agent to modulate the activity of a REEPand/or RTP protein can be confirmed in vivo, e.g., in an animal such asan animal model for a disease (e.g., an animal with an REEP and/or RTPrelated olfactory disorder).

B. Therapeutic Agents

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a REEP and/or RTP modulating agent or mimetic, a REEP and/or RTPspecific antibody, a REEP and/or RTP-binding partner, or an OR agonistor inhibitor) in an appropriate animal model (such as those describedherein) to determine the efficacy, toxicity, side effects, or mechanismof action, of treatment with such an agent. Furthermore, as describedabove, novel agents identified by the above-described screening assayscan be, e.g., used for treatments of olfactory disorders (e.g.,including, but not limited to, olfactory disorders).

IX. Pharmaceutical Compositions Containing REEP and RTP Nucleic Acid,Peptides, and Analogs

The present invention further provides pharmaceutical compositions whichmay comprise all or portions of REEP and/or RTP polynucleotidesequences, REEP and/or RTP polypeptides, inhibitors or antagonists ofREEP and/or RTP bioactivity, including antibodies, alone or incombination with at least one other agent, such as a stabilizingcompound, and may be administered in any sterile, biocompatiblepharmaceutical carrier, including, but not limited to, saline, bufferedsaline, dextrose, and water.

The methods of the present invention find use in treating diseases oraltering physiological states characterized by mutant REEP and/or RTPalleles (e.g., upper respiratory infections, tumors of the anteriorcranial fossa, and Kallmann syndrome, Foster Kennedy syndrome,Parkinson's disease, Alzheimer's disease, Huntington chorea). Peptidescan be administered to the patient intravenously in a pharmaceuticallyacceptable carrier such as physiological saline. Standard methods forintracellular delivery of peptides can be used (e.g., delivery vialiposome). Such methods are well known to those of ordinary skill in theart. The formulations of this invention are useful for parenteraladministration, such as intravenous, subcutaneous, intramuscular, andintraperitoneal. Therapeutic administration of a polypeptideintracellularly can also be accomplished using gene therapy as describedabove.

As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

Accordingly, in some embodiments of the present invention, REEP and/orRTP nucleotide and REEP and/or RTP amino acid sequences can beadministered to a patient alone, or in combination with other nucleotidesequences, drugs or hormones or in pharmaceutical compositions where itis mixed with excipient(s) or other pharmaceutically acceptablecarriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert. Inanother embodiment of the present invention, REEP and/or RTPpolynucleotide sequences or REEP and/or RTP amino acid sequences may beadministered alone to individuals subject to or suffering from adisease.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of REEP and/or RTP may be that amount that suppressesolfactory disorder related symptoms. Determination of effective amountsis well within the capability of those skilled in the art, especially inlight of the disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with a filler orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For polynucleotide or amino acid sequences of REEP and/orRTP, conditions indicated on the label may include treatment ofcondition related to olfactory disorders.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (particularly murine models) to achieve a desirable circulatingconcentration range that adjusts REEP and/or RTP levels.

A therapeutically effective dose refers to that amount of REEP and/orRTP that ameliorates symptoms of the disease state. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and additional animal studiescan be used in formulating a range of dosage for human use. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.01 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference). Those skilled in theart will employ different formulations for REEP and/or RTP than for theinhibitors of REEP and/or RTP. Administration to the bone marrow maynecessitate delivery in a manner different from intravenous injections.

X. RNA Interference (RNAi)

RNAi represents an evolutionary conserved cellular defense forcontrolling the expression of foreign genes in most eukaryotes,including humans. RNAi is triggered by double-stranded RNA (dsRNA) andcauses sequence-specific mRNA degradation of single-stranded target RNAshomologous in response to dsRNA. The mediators of mRNA degradation aresmall interfering RNA duplexes (siRNAs), which are normally producedfrom long dsRNA by enzymatic cleavage in the cell. siRNAs are generallyapproximately twenty-one nucleotides in length (e.g. 21-23 nucleotidesin length), and have a base-paired structure characterized by twonucleotide 3′-overhangs. Following the introduction of a small RNA, orRNAi, into the cell, it is believed the sequence is delivered to anenzyme complex called RISC(RNA-induced silencing complex). RISCrecognizes the target and cleaves it with an endonuclease. It is notedthat if larger RNA sequences are delivered to a cell, RNase III enzyme(Dicer) converts longer dsRNA into 21-23 nt ds siRNA fragments.

Chemically synthesized siRNAs have become powerful reagents forgenome-wide analysis of mammalian gene function in cultured somaticcells. Beyond their value for validation of gene function, siRNAs alsohold great potential as gene-specific therapeutic agents (Tuschl andBorkhardt, Molecular Intervent. 2002; 2(3):158-67, herein incorporatedby reference).

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference.

XI. RNAi for REEP and RTP

As discussed above, the present invention provides RNAi for inhibitingthe expression of the REEP and/or RTP polypeptide in cells, ORs, orpathway components involved in the expression or activity of suchcomponents.

A. Designing and Testing RNAi for REEP and/or RTP

In order to design siRNAs for REEP and/or RTP (e.g. that target REEPand/or RTP mRNA) software design tools are available in the art (e.g. onthe Internet). For example, Oligoengine's web page has one such designtool that finds RNAi candidates based on Elbashir's (Elbashir et al,Methods 2002; 26: 199-213, herein incorporated by reference) criteria.Other design tools may also be used, such as the Cenix Bioscience designtool offered by Ambion. In addition, there is also the Si2 silencingduplex offered by Oligoengine.

There are also RNA folding software programs available that allow one todetermine if the mRNA has a tendency to fold on its own and form a“hair-pin” (which in the case of dsRNAi is not as desirable since onegoal is to have the RNAi attach to the mRNA and not itself). Onepreferred configuration is an open configuration with three or lessbonds. Generally, a positive delta G is desirable to show that it wouldnot tend to fold on itself spontaneously.

siRNA candidate molecules that are generated can be, for example,screened in an animal model of an olfactory disorder for thequantitative evaluation of REEP and/or RTP expression in vivo usingsimilar techniques as described above.

B. Expression Cassettes

REEP and/or RTP specific siRNAs of the present invention may besynthesized chemically. Chemical synthesis can be achieved by any methodknown or discovered in the art. Alternatively, REEP and/or RTP specificsiRNAs of the present invention may be synthesized by methods whichcomprise synthesis by transcription. In some embodiments, transcriptionis in vitro, as from a DNA template and bacteriophage RNA polymerasepromoter, in other embodiments, synthesis is in vivo, as from a gene anda promoter. Separate-stranded duplex siRNA, where the two strands aresynthesized separately and annealed, can also be synthesized chemicallyby any method known or discovered in the art. Alternatively, ds siRNAare synthesized by methods that comprise synthesis by transcription. Insome embodiments, the two strands of the double-stranded region of asiRNA are expressed separately by two different expression cassettes,either in vitro (e.g., in a transcription system) or in vivo in a hostcell, and then brought together to form a duplex.

Thus, in another aspect, the present invention provides a compositioncomprising an expression cassette comprising a promoter and a gene thatencodes a siRNA specific for REEP and/or RTP. In some embodiments, thetranscribed siRNA forms a single strand of a separate-stranded duplex(or double-stranded, or ds) siRNA of about 18 to 25 base pairs long;thus, formation of ds siRNA requires transcription of each of the twodifferent strands of a ds siRNA. The term “gene” in the expressioncassette refers to a nucleic acid sequence that comprises codingsequences necessary for the production of a siRNA. Thus, a gene includesbut is not limited to coding sequences for a strand of a ds siRNA.

Generally, a DNA expression cassette comprises a chemically synthesizedor recombinant DNA molecule containing at least one gene, or desiredcoding sequence for a single strand of a ds siRNA, and appropriatenucleic acid sequences necessary for the expression of the operablylinked coding sequence, either in vitro or in vivo. Expression in vitromay include expression in transcription systems and intranscription/translation systems. Expression in vivo may includeexpression in a particular host cell and/or organism. Nucleic acidsequences necessary for expression in a prokaryotic cell or in aprokaryotic in vitro expression system are well known and usuallyinclude a promoter, an operator, and a ribosome binding site, oftenalong with other sequences. Eukaryotic in vitro transcription systemsand cells are known to utilize promoters, enhancers, and termination andpolyadenylation signals. Nucleic acid sequences necessary for expressionvia bacterial RNA polymerases (such as T3, T7, and SP6), referred to asa transcription template in the art, include a template DNA strand whichhas a polymerase promoter region followed by the complement of the RNAsequence desired (or the coding sequence or gene for the siRNA). Inorder to create a transcription template, a complementary strand isannealed to the promoter portion of the template strand.

In any of the expression cassettes described above, the gene may encodea transcript that contains at least one cleavage site, such that whencleaved results in at least two cleavage products. Such products caninclude the two opposite strands of a ds siRNA. In an expression systemfor expression in a eukaryotic cell, the promoter may be constitutive orinducible; the promoter may also be tissue or organ specific (e.g.specific to the eye), or specific to a developmental phase. Preferably,the promoter is positioned 5′ to the transcribed region. Other promotersare also contemplated; such promoters include other polymerase IIIpromoters and microRNA promoters.

Preferably, a eukaryotic expression cassette further comprises atranscription termination signal suitable for use with the promoter; forexample, when the promoter is recognized by RNA polymerase III, thetermination signal is an RNA polymerase III termination signal. Thecassette may also include sites for stable integration into a host cellgenome.

C. Vectors

In other aspects of the present invention, the compositions comprise avector comprising a gene encoding an siRNA specific for REEP and/or RTPor preferably at least one expression cassette comprising a promoter anda gene which encodes a sequence necessary for the production of a siRNAspecific for REEP and/or RTP (an siRNA gene). The vectors may furthercomprise marker genes, reporter genes, selection genes, or genes ofinterest, such as experimental genes. Vectors of the present inventioninclude cloning vectors and expression vectors. Expression vectors maybe used in in vitro transcription/translation systems, as well as in invivo in a host cell. Expression vectors used in vivo in a host cell maybe transfected into a host cell, either transiently, or stably. Thus, avector may also include sites for stable integration into a host cellgenome.

In some embodiments, it is useful to clone a siRNA gene downstream of abacteriophage RNA polymerase promoter into a multicopy plasmid. Avariety of transcription vectors containing bacteriophage RNA polymerasepromoters (such as T7 promoters) are available. Alternatively, DNAsynthesis can be used to add a bacteriophage RNA polymerase promoterupstream of a siRNA coding sequence. The cloned plasmid DNA, linearizedwith a restriction enzyme, can then be used as a transcription template(See for example Milligan, J F and Uhlenbeck, O C (1989) Methods inEnzymology 180: 51-64).

In other embodiments of the present invention, vectors include, but arenot limited to, chromosomal, nonchromosomal and synthetic DNA sequences(e.g., derivatives of viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies). It is contemplated that any vector may be usedas long as it is expressed in the appropriate system (either in vitro orin vivo) and viable in the host when used in vivo; these two criteriaare sufficient for transient transfection. For stable transfection, thevector is also replicable in the host.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. In some embodiments of the presentinvention, mammalian expression vectors comprise an origin ofreplication, suitable promoters and enhancers, and also any necessaryribosome binding sites, polyadenylation sites, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. In other embodiments, DNA sequences derivedfrom the SV40 splice, and polyadenylation sites may be used to providethe required non-transcribed genetic elements.

In certain embodiments of the present invention, a gene sequence in anexpression vector which is not part of an expression cassette comprisinga siRNA gene (specific for REEP1, RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C,RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6)) is operativelylinked to an appropriate expression control sequence(s) (promoter) todirect mRNA synthesis. In some embodiments, the gene sequence is amarker gene or a selection gene. Promoters useful in the presentinvention include, but are not limited to, the cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein promoters and other promoters known to controlexpression of gene in mammalian cells or their viruses. In otherembodiments of the present invention, recombinant expression vectorsinclude origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture).

In some embodiments of the present invention, transcription of DNAencoding a gene is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase its transcription.Enhancers useful in the present invention include, but are not limitedto, a cytomegalovirus early promoter enhancer, the polyoma enhancer onthe late side of the replication origin, and adenovirus enhancers.

Preferably the design of a vector is configured to deliver the RNAi formore permanent inhibition. For example the pSilencer siRNA expressionvector offered by Ambion, the pSuper RNAi system offered by Oligoengine,and the GneSilencer System offered by IMGENEX. These are all plasmidvector based RNAis. BD Biosciences offer the RNAi-Ready pSIREN Vectors,that allow both a Plasmid-based vectors and an Adenoviral or aRetroviral delivery formats. Ambion is expected to release an adenoviralvector for siRNA shortly. For the design of a vector there is nolimitation regarding the folding pattern since there is no concernregarding the formation of a hairpin or at least there are no studiesthat found any difference in performance related to the mRNA foldingpattern. Therefore, SEQ ID NOS: 1-20, for example, may be used with in aVector (both Plasmid and Viral) delivery systems.

It is noted that Ambion offers a design tool for a vector on their webpage, and BD Biosciences offers a manual for the design of a vector,both of which are useful for designing vectors for siRNA.

D. Transfecting Cells

In yet other aspects, the present invention provides compositionscomprising cells transfected by an expression cassette of the presentinvention as described above, or by a vector of the present invention,where the vector comprises an expression cassette (or simply the siRNAgene) of the present invention, as described above. In some embodimentsof the present invention, the host cell is a mammalian cell. Atransfected cell may be a cultured cell or a tissue, organ, ororganismal cell. Specific examples of cultured host cells include, butare not limited to, Chinese hamster ovary (CHO) cells, COS-7 lines ofmonkey kidney fibroblasts, 293T, C127, 3T3, HeLa, and BHK cell lines.Specific examples of host cells in vivo include tumor tissue and eyetissue.

The cells may be transfected transiently or stably (e.g. DNA expressingthe siRNA is stably integrated and expressed by the host cell's genome).The cells may also be transfected with an expression cassette of thepresent invention, or they are transfected with an expression vector ofthe present invention. In some embodiments, transfected cells arecultured mammalian cells, preferably human cells. In other embodiments,they are tissue, organ, or organismal cells.

In the present invention, cells to be transfected in vitro are typicallycultured prior to transfection according to methods which are well knownin the art, as for example by the preferred methods as defined by theAmerican Tissue Culture Collection. In certain embodiments of thepresent invention, cells are transfected with siRNAs that aresynthesized exogenously (or in vitro, as by chemical methods or in vitrotranscription methods), or they are transfected with expressioncassettes or vectors, which express siRNAs within the transfected cell.

In some embodiments, cells are transfected with siRNAs by any methodknown or discovered in the art which allows a cell to take up exogenousRNA and remain viable. Non-limiting examples include electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun, osmotic shock, temperatureshock, and electroporation, and pressure treatment. In alternative,embodiments, the siRNAs are introduced in vivo by lipofection, as hasbeen reported (as, for example, by Elbashir et al. (2001) Nature 411:494-498, herein incorporated by reference).

In other embodiments expression cassettes or vectors comprising at leastone expression cassette are introduced into the desired host cells bymethods known in the art, including but not limited to transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (See e.g., Wu et al. (1992) J. Biol. Chem.,267:963; Wu and Wu (1988) J. Biol. Chem., 263:14621; and Williams et al.(1991) Proc. Natl. Acad. Sci. USA 88:272). Receptor-mediated DNAdelivery approaches are also used (Curiel et al. (1992) Hum. Gene Ther.,3:147; and Wu and Wu (1987) J. Biol. Chem., 262:4429). In someembodiments, various methods are used to enhance transfection of thecells. These methods include but are not limited to osmotic shock,temperature shock, and electroporation, and pressure treatment.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker. The useof cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes. Particularly useful lipid compounds and compositions fortransfer of nucleic acids are described in WO95/18863 and WO96/17823,and in U.S. Pat. No. 5,459,127, herein incorporated by reference. Othermolecules are also useful for facilitating transfection of a nucleicacid in vivo, such as a cationic oligopeptide (e.g., WO95/21931),peptides derived from DNA binding proteins (e.g., WO96/25508), or acationic polymer (e.g., WO95/21931).

It is also possible to introduce a sequence encoding a siRNA in vivo asa naked DNA, either as an expression cassette or as a vector. Methodsfor formulating and administering naked DNA to mammalian muscle tissueare disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of whichare herein incorporated by reference.

Stable transfection typically requires the presence of a selectablemarker in the vector used for transfection. Transfected cells are thensubjected to a selection procedure. Generally, selection involvesgrowing the cells in a toxic substance, such as G418 or Hygromycin B,such that only those cells expressing a transfected marker geneconferring resistance to the toxic substance upon the transfected cellsurvive and grow. Such selection techniques are well known in the art.Typical selectable markers are well known, and include genes encodingresistance to G418 or hygromycin B.

In preferred embodiments, the transfecting agent is OLIGOFECTAMINE.OLIGOFECTAMINE is a lipid based transfection reagent. Additional exampleof lipid based transfection reagents that were designed for thetransfection of dsRNAis are the Transit-TKO reagent which is provided byMirus (Madison, Wis.) and the jetSI which was introduced byPolyplus-trasfection SAS. In addition, the Silencer siRNA TransfectionKit provided by Ambion's includes siPORT Amine and siPORT Lipidtransfection agents. Roche offers the Fugene 6 transfection reagentsthat are also lipid based. There is an option to use electroporation incell culture. Preferably a plasmid vector delivery system is transfectedinto the cell with OLIGOFECTAMINE provided by Invitrogen or with siPORTXP-1 transfection agent provided by Ambion.

In certain embodiments, certain chemical modifications of the dsRNAissuch as changing the lipophilicity of the molecule may be employed(e.g., attachment of lipophilic residues at the 3′ termini of thedsRNA). Delivery of dsRNAs into organisms may also be achieved withmethods previously developed for the application of antisenseoligonucleotides such as injection of liposomes-encapsulated molecules.

E. Kits

The present invention also provides kits comprising at least oneexpression cassette comprising a siRNA gene specific for REEP and/orRTP. In some aspects, a transcript from the expression cassette forms adouble stranded siRNA of about 18 to 25 base pairs long. In otherembodiments, the expression cassette is contained within a vector, asdescribed above, where the vector can be used in in vitro transcriptionor transcription/translation systems, or used in vivo to transfectcells, either transiently or stably.

In other aspects, the kit comprises at least two expression cassettes,each of which comprises a siRNA gene, such that at least one geneencodes one strand of a siRNA that combines with a strand encoded by asecond cassette to form a ds siRNA; the ds siRNA so produced is any ofthe embodiments described above. These cassettes may comprise a promoterand a sequence encoding one strand of a ds siRNA. In some furtherembodiments, the two expression cassettes are present in a singlevector; in other embodiments, the two expression cassettes are presentin two different vectors. A vector with at least one expressioncassette, or two different vectors, each comprising a single expressioncassette, can be used in in vitro transcription ortranscription/translation systems, or used in vivo to transfect cells,either transiently or stably.

In yet other aspects, the kit comprises at least one expressioncassettes which comprises a gene which encodes two separate strands of ads siRNA and a processing site between the sequences encoding eachstrand such that, when the gene is transcribed, the transcript isprocessed, such as by cleavage, to result in two separate strands whichcan combine to form a ds siRNA, as described above.

In some embodiments, the present invention provides kits comprising; a)a composition comprising small interfering RNA duplexes (siRNAs)configured to inhibit expression of the REEP and/or RTP protein, and b)printed material with instructions for employing the composition fortreating a target cell expressing REEP and/or RTP protein via expressionof REEP and/or RTP mRNA under conditions such that the REEP and/or RTPmRNA is cleaved or otherwise disabled. In certain embodiments, theprinted material comprises instructions for employing the compositionfor treating eye disease.

F. Generating REEP and/or RTP specific siRNA

The present invention also provides methods of synthesizing siRNAsspecific for REEP and/or RTP (e.g. human REEP and/or RTP) or specificfor mutant or wild type forms of REEP and/or RTP. The siRNAs may besynthesized in vitro or in vivo. In vitro synthesis includes chemicalsynthesis and synthesis by in vitro transcription. In vitrotranscription is achieved in a transcription system, as from abacteriophage RNA polymerase, or in a transcription/translation system,as from a eukaryotic RNA polymerase. In vivo synthesis occurs in atransfected host cell.

The siRNAs synthesized in vitro, either chemically or by transcription,are used to transfect cells. Therefore, the present invention alsoprovides methods of transfecting host cells with siRNAs synthesized invitro; in particular embodiments, the siRNAs are synthesized by in vitrotranscription. The present invention further provides methods ofsilencing the REEP and/or RTP gene in vivo by transfecting cells withsiRNAs synthesized in vitro. In other methods, the siRNAs is expressedin vitro in a transcription/translation system from an expressioncassette or expression vector, along with an expression vector encodingand expressing a reporter gene.

The present invention also provides methods of expressing siRNAs in vivoby transfecting cells with expression cassettes or vectors which directsynthesis of siRNAs in vivo. The present invention also provides methodsof silencing genes in vivo by transfecting cells with expressioncassettes or vectors that direct synthesis of siRNAs in vivo.

XII. Identification of Odorant Receptor Ligands

The present invention provides methods for identifying ligands specificfor odorant receptors. The present invention is not limited to aparticular method for identifying ligands specific for odorantreceptors. In preferred embodiments, the present invention provides acell line (e.g., heterologous 293T cell line) expressing an odorantreceptor of interest (e.g., any human odorant receptor) localized to thecell surface, REEP1, RTP1 or variant thereof, RTP2, and G_(αolf).Activation of an odorant receptor results in an increase in cAMP. Assuch, in some embodiments, the cell line further comprises a cAMPresponsive element linked with a reporting agent (e.g., luciferase) fordetecting odorant receptor activation. An odiferous molecule (e.g.,eugenol) is exposed to the cell line. If the odiferous molecule is aligand specific for the odorant receptor, luciferase expression or achange in luciferase expression is detectable (see, e.g., Example 7).

EXAMPLES

To identify accessory proteins that are involved in targeting ORs to thecell surface, genes were screened for inducing functional cell surfaceexpression of ORs in HEK293T (293T) cells. It was discovered REEP1,RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1,RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5),and RTP4-A1-D1 (Chimera 6), were discovered that promote cell surfaceexpression of ORs. These proteins are expressed by olfactory neurons,interact with OR proteins, and enhance responses to odorants whenco-expressed with ORs in 293T cells. Furthermore, this has allowedconstruction of a heterologous expression system to identify new ORsthat respond to aliphatic odorants.

Example 1 Identification of Odorant Receptor Accessory Proteins

After hypothesizing that mammalian ORs require accessory protein(s) forfunctional cell surface expression, a search was instituted fordetecting such molecule(s). Long-SAGE (serial analysis of geneexpression) libraries (see, e.g., Saha, S., et al., (2002) NatBiotechnol 20, 508-512; herein incorporated by reference in itsentirety) were constructed from single olfactory neurons as well asneurons from the vomeronasal organ and genes were collected that areexpressed by these neurons. To identify candidate genes expressed by theolfactory neurons Digital Differential Display (see, e.g.,http://www.ncbi.nlm.nih.gov/UniGene/info_ddd.shtml) was also used.Candidate genes were investigated for ORFs that encode membraneassociated proteins. Genes were selected with similarities to knownchaperones and cloned the cDNAs from olfactory epithelium cDNAs. ThemRNA expression of each gene was verified by in situ hybridization.After isolating and subcloning into mammalian expression vectors, eachcDNA together with a mouse OR (MOR203-1) tagged with a 20 N-terminalamino acids of rhodopsin (Rho-tag), was transfected into 293T cells.Measurements were made assessing whether these clones had any effect onthe cell-surface expression of ORs by staining living cells usingantibodies against the Rho-tag (see, e.g., Laird, D. W., and Molday, R.S. (1988) Invest Opthalmol Vis Sci 29, 419-428; herein incorporated byreference in its entirety). When MOR203-1 was transfected alone,antibody staining detected only faint cell-surface expression in lessthan 1% of the cells. A schematic diagram outlining the screeningprocedure utilized with the present invention is provided at FIG. 1.

Example 2 REEP and/or RTP Enhance Cell Surface Expression of ORs

Two unrelated clones (of 61 tested) enhanced both the number andstaining intensity of cell surface expression of MOR203-1 (see FIG. 2A).The proteins encoded by these clones were named REEP1, for ReceptorExpression Enhancing Protein 1 and RTP1, for Receptor TransportingProtein 1. Subsequently, RTP2 was found, a close relative of RTP1. RTP2also enhanced cell surface expression of MOR203-1. Next, REEP1, RTP1,and RTP2 were tested to detect a similar effect in promotingcell-surface expression of other ORs. Four different ORs (mouse OREG,mouse olfr62, mouse OR-S46 and rat I7) were expressed in 293T cells withor without REEP1, RTP1, or RTP2. Co-transfection of BFP or GFPdemonstrated that transfection efficiency was consistent (˜70%).Additionally, ORs transfected with REEP and/or RTP generated moreimmunofluorescent cells and stronger signals in positive cells comparedwith ORs without REEP and/or RTP (see FIG. 2A). The signal intensity andthe number of immunopositive cells varied when using different ORs ateach condition. For example, in the case of rat I7, the surfaceexpression was significantly lower than that of other ORs tested.Nonetheless, occasional immunopositive cells were observed only when theaccessory proteins were co-expressed. The effects of RTP1 or RTP2 wereconsistently more robust than that of REEP1. The enhancement ofcell-surface expression was specific for ORs and not for other GPCRs:neither REEP and/or RTP enhanced expression of the β2 adrenergicreceptor, mT2R5 (a mouse bitter taste receptor) (see, e.g.,Chandrashekar, J., et al. (2000) Cell 100, 703-711; herein incorporatedby reference in its entirety), or a V2R pheromone receptor (VR4) (see,e.g., Matsunami, H., and Buck, L. B. (1997) Cell 90, 775-784; hereinincorporated by reference in its entirety) (see FIG. 2A and FIG. 3).Finally, enhancement of cell-surface expression of MOR203-1 was notobserved when other members of the REEP and RTP families (REEP2 andRTP4) were co-expressed.

In order to quantify the numbers and intensity of immunopositive cells,Fluorescence-activated cell sorting (FACS) analysis was performed. Tomonitor transfection and staining efficiency, HA-tagged β2 adrenergicreceptor was used as a control. More cells were labelled and thefluorescent signal was higher when ORs were expressed with the accessoryproteins (see FIGS. 2B and 2C and FIG. 4).

Example 3 REEP and/or RTP Genes Encode Transmembrane Proteins

The REEP1 gene encodes a protein of 201 amino acids, containing twoputative transmembrane domains (see FIG. 5A). Immunostaining ofC-terminal tagged REEP1 protein indicate that the C-terminal end isextracellular. BLAST searches identified homologous genes in diverseeukaryotic species. REEP1 showed limited similarities with yeast YOP1,barley HVA22, and human DP1/TB2 (see FIG. 5B). YOP1 is implicated invesicular transport (see, e.g., Brands, A., and Ho, T. H. (2002) PlantPhysiol 130, 1121-1131; herein incorporated by reference in itsentirety). Expression of HVA22 is induced by abscisic acid and regulatedby various environmental stresses such as extreme temperatures ordehydration (see, e.g., Chen, C. N., et al. (2002) Plant Mol Biol 49,633-644; Shen, Q., et al. (1993) J Biol Chem 268, 23652-23660; eachherein incorporated by reference in their entireties). DP1/TB2 isencoded by a gene deleted in colon cancers (see, e.g., Kinzler, K. W.,et al. (1991) Science 253, 661-665; herein incorporated by reference inits entirety) and a mouse homolog of DPI (REEP5) is downregulated whenmast cells are triggered by IgE plus antigen (see, e.g., Prieschl, E.,et al. (1996) Gene 169, 215-218; herein incorporated by reference in itsentirety). In the mouse genome, REEP1 has at least 5 additionalhomologous genes (designated REEP2-6) (see FIG. 5B).

RTP1 and RTP2 genes encode proteins with 263 and 223 amino acids,respectively and share a 73% sequence identity in amino acid level (seeFIG. 5C). Neither protein appears to have a signal sequence but bothhave a single putative transmembrane domain located near the C-terminalend. Immunostaining of the C-terminal tagged RTP1 suggest thatC-terminal end is extracellular. BLAST searches of the mouse genomeidentified two additional members, RTP3 and RTP4. There were no obviousRTP homologs outside vertebrate species. Nevertheless, C. elegans ODR-4(see, e.g., Dwyer, N. D., et al. (1998) Cell 93, 455-466; hereinincorporated by reference in its entirety) appears to have the samemembrane topology as the RTPs.

Example 4 REEP and/or RTP Are Specifically Expressed in OlfactoryNeurons

Northern blot analysis with RNAs extracted from various mouse tissuesrevealed that REEP1 and especially RTP1 and RTP2 are most prominentlyexpressed in olfactory and vomeronasal organs. REEP1 RNA was alsodetected at significant levels in the brain (see FIG. 6A). Long exposurerevealed faint signals for RTP1 and RTP2 in the brain. Expression intestis was not observed, where a subset of ORs are expressed (see, e.g.,Parmentier, M., et al. (1992) Nature 355, 453-455; Spehr, M., et al.(2003) Science 299, 2054-2058; each herein incorporated by reference intheir entireties).

In the olfactory epithelium, REEP and/or RTP were expressed specificallyin olfactory neurons, which is evident from comparison with OMPexpression, a marker for mature olfactory sensory neurons (see FIG. 6B).To avoid cross hybridization between RTP1 and RTP2 RNA, which are 87%identical at nucleotide level across the coding sequence, non-homologous3′UTR regions as probes were used in addition to probes corresponding tothe open reading frames. The signals were identical. No expression ofother REEP or RTP genes was detected in olfactory neurons with theexception of RTP4 which was expressed at lower levels (see FIG. 6B).Finally, REEP1 was expressed by a subset of brain cells (see FIG. 6C).

Example 5 REEP1 and RTP1 can Interact with ORs

Given the ability of REEP and/or RTP to promote cell-surface expressionof ORs, it was hypothesized that they may also interact with ORproteins. This was assessed using co-immunoprecipitation assays.HA-tagged MOR203-1 and Flag-tagged REEP1, RTP1 or ICAP-1, a negativecontrol (see, e.g., Zawistowski, J. S., et al. (2002) Hum Mol Genet. 11,389-396; herein incorporated by reference in its entirety) weretransfected in 293T cells. After the cell extracts were precipitatedwith anti-Flag antibodies, proteins were eluted in SDS-sample buffer atroom temperature whereupon western blotting analysis was performed todetect the OR proteins. OR proteins were detected as high molecularweight bands after precipitation of REEP1 or RTP1 (see FIG. 7B, lanes 1and 2). The majority of a control GPCR, β2 adrenergic receptor, did notform high-molecular weight oligomers using these elution conditions.Similarly, when the HA-MOR203-1 proteins were precipitated, REEP1 orRTP1 proteins were co-precipitated whereas ICAP-1 was not detectable(see FIG. 7C, lanes 1, 2 and 3). The present invention is not limited toa particular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, these resultsindicate that REEP1 and RTP1 complex with ORs.

Based on the protein interaction, it was hypothesized that thefunctional expression of the accessory proteins might be regulated bythe OR proteins. When only C-terminal Flag-tagged RTP1 was transfectedinto 293T cells, little cell-surface signal was detected, indicatingthat the majority of RTP proteins was inside the cells. In contrast,co-transfection of RTP1 and OR greatly enhanced cell-surface RTP1 (seeFIG. 7D). The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, these results demonstratedmutual dependence of ORs and RTP1 for cell-surface expression andindicated that effective cell surface expression of the both ORs andRTP1 requires the formation of a relatively stable receptor complexbetween the two. When the C-terminal tagged REEP1 was expressed, a smallamount of cell-surface REEP1 was observed. Unlike RTP1, co-expression ofthe OR proteins did not facilitate cell-surface expression of REEP1 (seeFIG. 7E).

Example 6 REEP and/or RTP Enhance OR Function

Poor odorant evoked signaling activity in heterologous cell culturesystems expressing ORs has been attributed to the poor cell surfaceexpression of ORs. The identification of REEP and/or RTP allowed adirect assessment of this issue. A luciferase reporter gene assay wasemployed in which a cAMP responsive element (CRE) mediated luciferasegene expression (see FIG. 8A). Because OR activation leads to anincrease in cAMP, activation of the mouse odorant receptor OREG by itsligand eugenol was measured in the presence and absence of REEP and/orRTP (see, e.g., Kajiya, K., et al. (2001) J Neurosci 21, 6018-6025;Touhara, K., et al., (1999) Proc Natl Acad Sci USA 96, 4040-4045; eachherein incorporated by reference in their entireties). As reportedpreviously, eugenol increased levels of OREG dependent luciferaseactivity (see, e.g., Katada, S., et al. (2003) Biochem Biophys ResCommun 305, 964-969; herein incorporated by reference in its entirety).Co-expression of OREG with REEP and/or RTP markedly enhancedodorant-dependent luciferase activity (FIG. 8B). Similar results wereobtained when vanillin or ethyl vanillin, two other OREG ligands wereapplied. Since RTP4 is also expressed at low levels in olfactoryepithelium, this protein was co-expressed with OREG, but this did notproduce a significant increase in luciferase reporter gene activity.

Other GPCRs can exhibit change in ligand specificity depending onaccessory proteins (see, e.g., McLatchie, L. M., et al. (1998) Nature393, 333-339; herein incorporated by reference in its entirety).(McLatchie et al., 1998). To investigate whether REEP1, RTP1, or RTP2alter the ligand selectivity of ORs, OREG and OR-S46 with their agonistsand related chemicals were tested. No substantial changes in relativechemical selectivity were observed when the receptors were co-expressedwith the accessory proteins (see FIG. 8C).

Example 7 Constructing a Functional Assay to Identify Odorant-ReceptorInteractions

To facilitate analysis of odorant-OR interactions, 293T cell lines wereestablished which stably express REEP1, RTP1, RTP2 and G_(αolf), the Gprotein alpha subunit that couples to OR (see, e.g., Belluscio, L., etal. (1998) Neuron 20, 69-81; Jones, D. T., and Reed, R. R. (1989).Science 244, 790-795; each herein incorporated by reference in theirentireties). To establish such cells, linearized expression vectorscontaining mouse REEP1, RTP1, RTP2 and G_(αolf) ORFs were transfectedinto 293T cells with PGK-Pac (puromycin resistant gene) (see, e.g.,Watanabe, S., et al. (1995) Biochem Biophys Res Commun 213, 130-137;herein incorporated by reference in its entirety). Among the puromycinresistant clones, clone 3A showed a large response to eugenol when OREGwas transfected, and was named Hana3A. RT-PCR analysis indicated thatHana3A cells express exogenous REEP1, RTP1, RTP2 and G_(αolf) (see FIG.9). Enhanced cell-surface expression was observed when OREG or other ORswere transfected in Hana3A cells and immunostained (see FIG. 10). Totest whether Hana3A cells also increased the ligand response in theluciferase assay, the CRE-luciferase reporter gene along with eitherOREG (HA-tagged), OR-S46 or OR-S50 were co-transfected and stimulatedthe cells with their ligands, eugenol, nonanoic acid, and nonanedioicacid, respectively (see, e.g., Malnic, B., et al. (1999) Cell 96,713-723; Touhara, K., et al. (1999) Proc Natl Acad Sci USA 96,4040-4045; each herein incorporated by reference in their entireties).Little luciferase induction was observed when HA-OREG was expressed in293T cells. In contrast, when Hana3A cells were used, an enhancement inluciferase activity was observed following eugenol stimulation (see FIG.8D). Similar results were obtained using two additional ORs, OR-S46 andOR-S50. The OR-S50 gene did not produce a luciferase response in 293Tcells, whereas the same receptor transfected into the Hana3A cellsproduced robust luciferase activity (see FIG. 8D). Expression ofG_(αolf) alone in 293T cells had little or no effect on OR activationusing this assay.

In order to confirm the increased OR function in the presence of REEP1,RTP1 and RTP2, the amount of cAMP upon ligand stimulation was measuredusing 293T cells expressing G_(αolf) and Hana3A cells. When OREG wastransfected and eugenol was added to stimulate the OR, more cAMP wasproduced in Hana3A cells. In contrast, when the β2 adrenergic receptorwas expressed and isoproterenol was used, no significant differences incAMP production were observed (see FIG. 8E). The present invention isnot limited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, this further supports a specific role of the accessoryproteins in functional OR expression.

Previous studies demonstrated that single olfactory neurons that areactivated by aliphatic alcohols and acids express specific ORs,primarily class I (fish-like) ORs (see, e.g., Malnic, B., et al. (1999)Cell 96, 713-723; Zhang, X., and Firestein, S. (2002) Nat Neurosci 5,124-133; each herein incorporated by reference in their entireties).Four ORs (S6/79, S18, S46 and S50) previously assayed using othertechniques (see, e.g., Malnic, B., et al. (1999) Cell 96, 713-723;herein incorporated by reference in its entirety) were tested against anassay panel of aliphatic alcohols, aldehydes and acids and some otherodorants. Additionally, five “orphan” class I ORs (MOR23-1, MOR31-4,MOR31-6, MOR32-5 and MOR32-11) whose cognate ligands were unknown weretested. At a suprathreshold concentration of 100 uM, all these ORs wereodorant selective, responding to only a small subset of the odorantstested (see FIG. 11A). This specificity was retained at lower, morephysiologically relevant concentrations. Many of these ORs responded toodorants present in micromolar concentrations. The cell-surfaceexpression of these ORs by living-cell immunofluorescence wereevaluated. Some ORs (S18, MOR31-4, MOR31-6 and MOR32-5) were stronglyexpressed while other ORs (S6, S50, MOR23-1, MOR32-11) were weaklyexpressed (see FIG. 12). The present invention is not limited to aparticular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, these resultssuggest that weak expression was sufficient to produce a significantresponse to odorants at physiologically relevant concentrations.Finally, two additional orphan class II ORs, MOR203-1 and olfr62, weretested against a panel of 139 odorants. MOR203-1 responded to highconcentrations of nonanoic acid (see FIG. 11B). Olfr62 responded tocoumarine and piperonal (see FIG. 11C). Several related aromaticcompounds were next tested and 2-coumaranone was identified as apreferred ligand for olfr62 (see FIG. 11C). When parental 293T cells forthese ORs were used in this luciferase assay, little or no response tothe odorants was observed. The present invention is not limited to aparticular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, these resultsfurther demonstrate the importance of REEP and/or RTP in functional ORexpression.

Example 8 REEP and/or RTP Function During Receptor Folding, Transport,and/or Odorant Recognition

Expression of GPCRs is a complex process that includes protein folding,post-translational modifications and transport through cellularcompartments including the ER and Golgi apparatus. Additionally,evidence indicates that the proper targeting of GPCRs to the plasmamembrane may involve homo or heterodimerization (see, e.g., Angers, S.,et al. (2002) Annu Rev Pharmacol Toxicol 42, 409-435; hereinincorporated by reference in its entirety). The present invention is notlimited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, though REEP and/or RTP can function at any of these stepsof OR expression, three possibilities are presented in FIG. 13 regardingtheir possible interaction.

First, REEP and/or RTP promote correct folding of ORs in the ER. NinaA,a cyclophilin homolog of Drosophila, was identified as a chaperoneprotein for rhodopsin and thought to facilitate the correct folding(see, e.g., Baker, E. K., et al. (1994) Embo J 13, 4886-4895; Shieh, B.H., et al. (1989) Nature 338, 67-70; each herein incorporated byreference in their entireties). The plant homologs of REEP1, HVA22s, arestress-induced genes and may allow plants to tolerate adverse conditions(see, e.g., Chen, C. N., et al. (2002) Plant Mol Biol 49, 633-644; Shen,Q., et al. (1993) J Biol Chem 268, 23652-23660; each herein incorporatedby reference in their entireties). The present invention is not limitedto a particular mechanism. Indeed, an understanding of the mechanism isnot necessary to practice the present invention. Nonetheless, while theprecise roles of HVA22s are not known, since a number of stress inducedproteins, such as heat shock proteins, function as chaperones, it isconceivable that HVA22s and, by analogy, perhaps REEP1 act as chaperonesto promote folding.

Second, REEP1, RTP1, and RTP2 facilitate the transport of specificvesicles/cargos that include ORs. Consistent with this idea, a REEP1homolog in yeast, YOP1P, has been implicated in Rab-mediated vesicletransport (see, e.g., Brands, A., and Ho, T. H. (2002) Plant Physiol130, 1121-1131; Calero, M. (2001) J Biol Chem 276, 12100-12112; eachherein incorporated by reference in their entireties). In C. elegans, aclathrin adaptor subunit, UNC-101, mediates trafficking of chemosensoryreceptors to olfactory cilia (see, e.g., Dwyer, N. D., et al., (2001)Neuron 31, 277-287; herein incorporated by reference in its entirety).

Third, REEP1, RTP1, and RTP2 act as a co-receptor with ORs. As shown inFIG. 7D, RTP1 cell surface expression is enhanced by co-expression ofORs. ORs may contain ER retention signal(s) that are masked by theassociation with RTPs (or REEP1), a mechanism similar to the regulationof cell-surface expression of GABA(B)R1 receptor by the association ofGABA(B)R2 (see, e.g., Jones, K. A., et al. (1998) Nature 396, 674-679;Kaupmann, K., et al. (1998) Nature 396, 683-687; White, J. H., et al.(1998) Nature 396, 679-682; each herein incorporated by reference intheir entireties). The REEPs and RTPs may have different orcomplementary roles, a hypothesis that is consistent with the absence ofany amino acid sequence similarity or specific sequence motifs.

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, the three roles outlined above arereasonable for REEP1, RTP1 and RTP2; however, other possible functionsare not excluded. Even though changes in ligand specificity of OREG orOR-S46 was not observed when expressed with REEP1, RTP1 or RTP2, it ispossible that they do play a role in modulating recognition profiles ofsome ORs. For example, different RAMP members change the ligandspecificity of calcitonin receptor like receptor (CRLR), a member ofGPCRs. CRLR expressed with RAMP 1 function as a CGRP receptor, whereasCRLR expressed with RAMP2 functions as adrenomedullin receptor (see,e.g., McLatchie, L. M., et al. (1998) Nature 393, 333-339; hereinincorporated by reference in its entirety).

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, many GPCRs, including V1R pheromonereceptors (see, e.g., Dulac, C., and Axel, R. (1995) Cell 83, 195-206;herein incorporated by reference in its entirety), T2R taste receptors(see, e.g., Adler et al. (2000) Cell 100, 693-702; Matsunami, H.,Montmayeur, J. P., and Buck, L. B. (2000) Nature 404, 601-604; eachherein incorporated by reference in their entireties), the α2Cadrenergic receptor (see, e.g., Hurt, C. M., et al. (2000) J Biol Chem275, 35424-35431; herein incorporated by reference in its entirety), andthe thyrotropin-releasing hormone receptor (see, e.g., Yu, R., andHinkle, P. M. (1997) Mol Pharmacol 51, 785-793; herein incorporated byreference in its entirety), appear to require cofactor(s) for their cellsurface expression. Thus, REEP and RTP members may regulate traffickingof such GPCRs. In situ hybridization analysis has shown that REEP3,REEP5, RTP1 and RTP2 are all expressed by the VNO neurons. In addition,REEP members are differentially expressed in subset of brain cells (M.M.and H.M., unpublished observations). The strategy to create a list ofgenes expressed in specific cell types using SAGE and/or DigitalDifferential Display and screen genes that promote cell-surfaceexpression of the receptors could be applied in such cases.

Example 9 REEP and/or RTP Enable Investigations of OdorantReceptor-Odorant Interactions

An expression system has been established that permits rapididentification of ligands for ORs. This system was tested with twelveORs. Four of the tested ORs (S6/S79, S18, S46, and S50) were expressedin single olfactory neurons responding to aliphatic odorants (see, e.g.,Malnic, B., et al. (1999) Cell 96, 713-723; herein incorporated byreference in its entirety). The response profiles of OR-S50, but notthat of OR-S18 agreed with the previous report (see, e.g., Malnic, B.,et al. (1999) Cell 96, 713-723; herein incorporated by reference in itsentirety). In previous studies, olfactory neurons S6 and S79 expressedthe same OR(OR-S6/S79) and both responded to nonanedioic acid, althoughonly the olfactory neuron S79 responded to two odorants, heptanoic acidand octanoic acid (see, e.g., Malnic, B., et al. (1999) Cell 96,713-723; herein incorporated by reference in its entirety). Inexperiments conducted during the course of the present invention,OR-S6/S79 responded to nonanedioic acid but not to heptanoic acid oroctanoic acid. The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, these results support theolfactory neuron S6 response profile. Differences may be due to thevariation of responses when recording from single olfactory neurons.When multiple single olfactory neurons that expressed the same OR wererecorded against the same set of odorants using calcium imaging, theirresponse profiles were similar but different (see, e.g., Bozza, T., etal. (2002) J Neurosci 22, 3033-3043; herein incorporated by reference inits entirety).

Seven new ORs were identified that responded to different odorants inthe test panels. The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, these results demonstratethe applicability of this system to decode the ligand specificity ofORs. The profiles of the ORs in response to various odorants areconsistent with the idea of “combinatorial receptor code” where one ORresponds to multiple related odorants and one odorant activates multiplereceptors (see, e.g., Kajiya, K., et al. (2001) J Neurosci 21,6018-6025; Malnic, B., et al. Cell 96, 713-723; each herein incorporatedby reference in their entireties).

In experiments conducted during the course of the present invention, notonly three class I ORs (S46, MOR23-1, MOR31-4) but also MOR203-1, aclass II OR, responded to nonanoic acid. The present invention is notlimited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, these results indicate that very different ORs can respondto the same chemical, as MOR203-1 and other nonanoic acid ORs (MOR23-1,MOR31-4 and S46) are only 29-32% identical. Olfr62 is one of the closelyrelated ORs located near or at the IVA locus, implicated in isovalericacid sensation (see, e.g., Griff, I. C., and Reed, R. R. (1995) Cell 83,407-414; Zhang, X., and Firestein, S. (2002) Nat Neurosci 5, 124-133;each herein incorporated by reference in their entireties). Inexperiments conducted during the course of the present invention, olfr62did not respond to isovaleric acid but responded to coumarin and otherrelated aromatic compounds (see FIG. 11C). Eight other ORs located nearthe IVA locus were also tested, but none of them responded to isovalericacid. The present invention is not limited to a particular mechanism.Indeed, an understanding of the mechanism is not necessary to practicethe present invention. Nonetheless, these results suggest that these ORsare not involved in isovaleric acid detection.

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, the functional OR expression systemtogether with the annotation of virtually all the ORs in the mouse andhuman genomes (see, e.g., Glusman, G., et al. (2001) Genome Res 11,685-702; Young, J. M., et al. (2002) Hum Mol Genet. 11, 535-546; Zhang,X., and Firestein, S. (2002) Nat Neurosci 5, 124-133; Zozulya, S., etal. (2001) Genome Biol 2, 18; each herein incorporated by reference intheir entireties), provide a platform to investigate mammalianOR-odorant interaction in a comprehensive manner.

Example 10 Single-Cell Long SAGE Analysis

Single-cell RT-PCR was conducted as described (see, e.g., Brady, G., andIscove, N. N. (1993) Methods Enzymol 225, 611-623; Dulac, C., and Axel,R. (1995) Cell 83, 195-206; Matsunami, H., and Buck, L. B. (1997) Cell90, 775-784; each herein incorporated by reference in their entireties)with modifications. Briefly, adult mouse olfactory tissues weredissociated with dispase (Invitrogen) and collagenase (Invitrogen).Single cells were picked under inverted microscope usingmicromanipulator and transferred into 4.75 ul of lysis mix (1×PCR buffer(Roche), 1.5 mM MgCl2, 50 uM dNTPs, 200 ng/mg anchor primer(biotin-TATAGAATTCGCGGCCGCTCGCGA (T) 24), 0.3 U/ul Prime RNase Inhibitor(Eppendorf), and 0.4 U/ul rRNasin (Promega). PCR tubes containing lysedcells were heated to 65 degrees C. for 1 min, cooled at 4 degrees C. and0.25 ul of RT mix (170 U/ul Superscript II (Invitrogen), 35 U/ul PrimeRNase Inhibitor and 45 U/ul rRNasin.) was added and incubated at 37degrees C. for 10 min then 65 degrees C. for 10 min. 5 ul of TdT mix(1×PCR buffer (Roche), 1.5 mM MgCl2, 3 mM dATP, 1.25 U/ul TdT (Roche),0.05 U RNase H (Roche)) was added to each tube and incubated at 37degrees C. for 20 min then at 65 degrees C. for 10 min. 5 ul of theproduct was added to 50 ul of PCR mix (1×EX Taq buffer (Takara), 0.25 mMdNTPs, 20 ng/ul anchor primer, 2.5 U EX Taq HS polymerase (Takara)) andincubated at 95 degrees C. for 2 min, 37 degrees C. 5 min, 72 degrees C.20 min, then 28 cycles of 95 degrees C. 30 sec, 67 degrees C. 1 min, 72degrees C. 6 min plus 6 sec extension for each cycle, then 72 degrees C.for 10 min. Contents of amplified PCR products were analyzed using longSAGE protocols (see, e.g., Saha, S., et al. (2002) Nat Biotechnol 20,508-512; herein incorporated by reference in its entirety). Briefly,single-cell PCR products were cut with NlaIII (NEB). After biotinylatedDNA was bound to streptavidin magnetic beads (Dynal), linkers wereligated. The ligated DNA was cut by MmeI (NEB). The cleaved tags wereligated to form ditags and amplified by PCR. The PCR product was cutwith NlaIII and the ditags were ligated to form concatemers. They wereligated into pZero-1 vector (Invitrogen) and transformed. Singlecolonies were picked and sequenced. Tag sequences were analyzed usingSAGE2002 software and NCBI Blast searches.

Example 11 Vector Construction

cDNAs were amplified from olfactory epithelium cDNA using HotstarTaq DNApolymerase (Qiagen) or KOD DNA polymerase (Toyobo/Novagen) and subclonedinto pCI expression vectors (Promega). OR open reading frames wereamplified from genomic DNA of C57BL6 (MOR203-1 and S46), 129 (S18) orDBA2 (olfr62, S6/S79, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 andMOR32-11) and subcloned into pCI containing Rho-tag.

Example 12 Cell Culture and Immunocytochemistry

293T cells were maintained in minimal essential medium containing 10%fetal bovine serum (M10). Lipofectamine 2000 (Invitrogen) was used fortransfection. In live-cell staining, 16 hours after transfection, cellswere incubated in M10 containing anti rhodopsin antibody, 4D2 (see,e.g., Laird, D. W., and Molday, R. S. (1988) Invest Opthalmol V is Sci29, 419-428; herein incorporated by reference in its entirety) and 15 mMNaN₃ at 4 degrees C. for 1 hour. After washing, cells were incubatedwith Cy₃-conjugated anti mouse IgG (Jackson Immunologicals), washed andmounted. For FACS analysis, 4D2 and PE-conjugated anti mouse IgG(Jackson Immunologicals) were used to monitor the Rho tagged receptorexpression. Anti HA rabbit antibodies (Sigma) and Alexa 488 conjugatedanti rabbit IgG (Molecular Probes) was used to stain the HA-β2adrenergic receptor. To establish the Hana3a cells, 1 ug/ml of puromycinwas used for selection. 96 colonies were picked and assayed usingluciferase assay using OREG.

Example 13 Analysis of REEPs and RTPs

For prediction of signal peptide and transmembrane regions of REEPs andRTPs, SignalP (see, e.g., Nielsen, H., et al. (1997) Protein Eng 10,1-6; herein incorporated by reference in its entirety) was used andTMHMM, respectively. In order to create a phylogenetic tree, ClustalWwas used.

Example 14 Northern and In Situ Hybridization

Total RNAs from various tissues were extracted using Trizol reagent(Invitrogen) or Aurum Total RNA (Biorad). RNAs were electrophoresed onformaldehyde-agarose gel and transferred onto HybondN membrane(Amersham). Dig-labeled probes were hybridized to the membrane in Digeasyhyb solution (Roche) at 65 degrees C. After washing, anti-Dig AP(Roche) were applied and the membranes were washed. The signals weredetected with CDP-Star (Roche). The same membrane for all three probeswas used. In situ hybridization was performed as described (see, e.g.,Matsunami, H., and Buck, L. B. (1997) Cell 90, 775-784; Matsunami, H.,et al. (2000) Nature 404, 601-604; Schaeren-Wiemers, N., andGerfin-Moser, A. (1993) Histochemistry 100, 431-440; each hereinincorporated by reference in their entireties). Briefly, Dig-labeled RNAprobes were hybridized with fresh frozen sections of three weeks oldCD-1 mice. After washing, Dig probes were reacted with anti-Dig AP andsignals were detected using NBT-BCIP.

Example 15 Immunoprecipitation

293T cells in 100 mm dishes were transfected with ORs, REEP1, and/orRTP1 cDNAs. 16 hours after transfection, cells were lysed in lysisbuffer (50 mM Tris (7.4), 150 mM NaCl, 1% NP-40, 0.5 mM PMSF, 2 mMBenzamidene, 0.5 ug/ml Leupeptin, 1.4 ug/ml pepstatin A, 2.4 ug/mlchymostatin, 15 ug/ml aprotinin, 1 mM sodium orthovanadate). The lysiswere incubated with anti-Flag M2 affinity gel (Sigma) or anti-HAaffinity matrix (Roche) for 2 hours at 4 degrees C. and washed withlysis buffer. Subsequently, the bound proteins were eluted by incubationwith SDS sample buffer at room temperature for 2 hours. SDS-PAGE andwestern blotting were performed according to Mini-Protean 3 Cell(Bio-Rad) instruction manual. ECL (Amersham) was used for detectingproteins on membranes.

Example 16 Luciferase Assay

Dual-Glo system (Promega) for luciferase assay was used. CRE-Luciferase(Stratagene) was used to measure the receptor activities. Renillaluciferase driven by constitutively active SV40 promoter (pRL-SV40:Promega) was used as an internal control. Cells were plated onpoly-D-lysine coated 96 well plates (BIOCOAT, Beckton Dickinson). After8 hours (for experiments shown in FIGS. 8B and 8C) or 12 hours (forexperiments shown in FIG. 8D and FIG. 11) after transfection, the mediumwas replaced with CD293 chemically defined medium (Invitrogen) and theplates were incubated for one hour at 37 degrees C. The medium wasreplaced with 50 ul of odorant solutions dissolved in CD293 andincubated for 110 hours (for experiments shown in FIGS. 8B and 8C) or 4hours (for experiments shown in FIG. 8D and FIG. 11) at 37 degrees C.The manufacture's protocol for measuring luciferase and Renillaluciferase activities was followed. Luminescence was measured usingWallac Victor 1420 (Perkin-Elmer). Normalized luciferase activity wascalculated as [Luc (N)-Luc (O)]/RL (N), where Luc (N)=Luminescent countof a certain well, Luc (O)=Luminescent count without odorant for eachOR, and RL (N)=Luminescent count of Renilla Luciferase of each well. ForcAMP assays, cells were plated onto 24-well plate. OREG or OREG/GolfcDNA was transfected into Hana3a or HEK293-Tcells, respectively. 14hours after transfection, the cells were incubated in CD293 for 2 hrs,and exposed to eugenol or isoproterenol in MEM containing 10 mM Hepesand 500 uM IBMX for 5 min. cAMP-Screen Direct System (AppliedBiosystems) was used to measure the cAMP levels. Prism software(Graphpad) was used for data analysis.

Example 17 Chemicals

All odorants were purchased from Sigma except octanoic acid fromCalbiochem. The chemicals used in finding cognate ligands for MOR203-1and olfr62 are provided in Example 19.

Example 18 Genbank Accession Numbers

The genbank accession numbers of mouse and human REEP1-6 and RTP1-4:AY562225-AY562244.

Example 19 Supplemental Materials

Chemicals that are used for initial ligand screening for MOR203-1 andolfr62 are the following: 1 (+)-Carvone, 2 L-Canvone, 3 (−)-Fenchone, 4Citral, 5 (1R)-(−)-Fenchone, 6 (+)-Fenchone, 7 Rosemary oil, 8 (−)-Roseoxide, 9 (+)-Rose oxide, 10 (−)-Camphor, 11 (S)-(−)-Limonene, 12(R)-(+)-1-Phenylethanol, 13 (S)-(+)-2-Phenylbutyric acid, 14(R)-(−)-2-Phenylbutyric acid, 15 2-Hexanone, 16 1-Pentanol, 171-Heptanol, 18 (±)-2-Butanol, 19 1-propanol, 20 1-Hexanol, 21(−)-Menthol, 22 (R)-(−)-2-Heptanol, 23 (−)-α-Terpineol, 24 (+)-Menthol,25 2-methyl-2-heptanol, 26 (S)-(+)-2-Octanol, 27 (S)-(+)-2-Butanol, 28(S)-(+)-2-Heptanol, 29 (R)-(−)-2-Octanolor P(+)-2 Octanol, 30 1-Decanol,31 (−)-β-Citronellol, 32 (S)-(−)-1-Phenylethanol, 33 Propionaldehyde, 34Undecanal, 35 Octanal (Caprylic aldehyde), 36 trans-Cinnamaldehyde, 37Nonanal (Pelargonaldehyde), 38 Heptaldehyde, 39 Decanal, 40 Hexanoicacid, 41 Hexanoic acid, 42 Heptanoic acid (Oenanthic acid), 43 Pentanoicacid, 44 Propionic acid, 45 Butyric acid, 46 Nonanoic acid, 47 Methylpropionate, 48 Ethyl butyrate, 49 Butyl butyrate, 50 tert-Butylpropionate, 51 Methyl butyrate, 52 Propyl butyrate, 53 Pentyl acetate,54 Dimethylpyrazine, 55 Isobutylamine, 56 Geraniol, 57 2-Pentanone, 582-Butanone, 59 (1S)-(−)-α-Pinene, 60 1,4-Cineole, 61 Phenetole, 62 Butylmethyl ether, 63 (R)-(+)-Pulegone, 64 Benzene, 65 Benzyl alcohol, 66Guaiacol, 67 Isopentylamine, 68 g-Caprolactone, 69 g-Caprolactone, 70octen, 71 Allyl heptanoate, 72 a-Amylcinnamaldehyde, 73 Amyl hexanoate,74 amylbutyrate, 75 Anethole, 76 Anisaldehyde, 77 Benzophenone, 78Benzyl acetate, 79 Benzyl salicylate, 80 Butyl heptanoate, 81 camphor((+)-Camphor), 82 Cedryl acetate, 83 Cinnamyl alcohol, 84Cinnamaldehyde, 85 (R)-(+)-Citronellal, 86 (S)-(−)-Citronellal, 87citronellol, 88 Coumarin, 89 Cyclohexanone, 90 p-cymene, 915,5-Dimethyl-1,3-cyclohexanedione (Dimedone), 92 ethylamylketone(3-Octanone), 93 Eucalyptol, 94 Heptyl isobutyrate, 95 Hexyl acetate, 96a-Hexylcinnamaldehyde, 97 Isobornyl acetate, 98 Linalool, 99 Lyral(a-Amylcinnamaldehyde dimethyl acetal), 100 Hydroxycitronellal, 101p-Tolyl isobutyrate, 102 o-Tolyl isobutyrate, 103 p-Tolyl phenylacetate,104 2-Methoxy-3-Methyl-pyrazine, 105 2-Methoxypyrazine, 106 Methylsalicylate, 107 Myrcene, 108 w-Pentadecalactone, 109 prenylacetate, 1102-Phenylethanol, 111 2-Phenethyl acetate, 112 Piperonal, 113 Pyrazine,114 Sassafras oil, 115 thymol, 116 Triethylamine, 117 2-Heptanone, 118Methyl eugenol, 119 eugenol, 120 Eugenol methyl ether, 121Butyraldehyde, 122 Hexanal, 123 1-Pentanol, 124 valeraldehyde, 125Azelaic acid dichloride, 126 Azelaic acid, 127 Isovaleric acid, 128Decanoic acid, 129 Vanillic acid, 130 1-Octanol, 131 4-Ethylphenol, 132Heptaldehyde, 133 1-Nonanol, 134 Nonanal, 135 Ethyl vanillin, 136Vanillin, 137 Acetophenone, 138 2-Ethylphenol, 139 Octanal.

Chemicals related to coumarin and piperonal (used for olfr62) are thefollowing: 140 Benzaldehyde, 141 Piperonyl alcohol, 1424-Hydroxycoumarin, 143 4-Chromanone, 144 2-Coumaranone.

Example 20 Activation Patterns of Human Odorant Receptors

Hana3A cells (293T cells expressing mouse REEP1, RTP1, RTP2, andG_(αolf)) were used. CRE-Luciferase (Stratagene) was used to measureodorant receptor activities. The following human odorant receptors weretested for expression patterns in response to various odiferous agents:36, 35, 11, 57, 58, 9, 3, 42, 81, 82, 66, 13, 87, 33, 44, 43, 77, 75,64, 59, 12, 62, 60, 120, 90, 95, 160, and 106. The following odiferousagents were used to test human odorant receptor expression patterns:Pyridine, 2,2′-(Dithiodimethylene)difuran, 1-Decanol, 1-Hexanol,(−)-Fenchone, (+)-Fenchone, Geraniol, 2-Pentanone, Benzyl salicylate,(+)-Menthol, (−)-Menthol, Benzene, Undecanal, Methyl butylate, Heptylisobutyrate, p-Tolyl isobutyrate, amylbutyrate, Ethyl butyrate, Hexylacetate, Pentyl Acetate, Piperonyl acetate, (−)-b-Citronellol,citronellol, Eugenol methyl ether, Methyl Eugenol, a-Amylcinnamaldehyde,a-Amylcinnamaldehyde dimethyl acetal, Cinnamaldehyde,a-Hexylcinnamaldehyde, Hydroxycitronnellal, Citral, (R)-(+)-Citronellal,(S)-(−)-Citronellal, p-Toly phenylacetate, Allyl phenylacetate,Propionic acid, Azelaic acid dichloride, isovaleric acid,(R)-(−)-2-Phenylbutyric acid, (S)-(+)-2-Phenylbutyric acid, HeptanoicAcid, Octanoic Acid, Valeric Acid, Hexanoic Acid, and Butyl butyrate.Renilla luciferase driven by constitutively active SV40 promoter(pRL-SV40: Promega) was used as an internal control. Dual-Glo system(Promega) was used for the luciferase assay. Cells were plated onpoly-D-lysine coated 96 well plates (BIOCOAT, Beckton Dickinson). 12-16hours after transfection, the medium was replaced with CD293 chemicallydefined medium (Invitrogen) and the plates were incubated for one hourat 37 degrees C. The medium was replaced with 50 μl of odorant solutionsdissolved in CD293 and incubated for 4 hours at 37 degrees C. Themanufacture's protocol was followed for measuring luciferase and Renillaluciferase activities. Luminescence was measured using Wallac Victor1420 (Perkin-Elmer). Normalized luciferase activity was calculated as[Luc (N)−Luc (0)]/RL (N), where Luc (N)=Luminescent count of a certainwell, Luc (0)=Luminescent count without odorant for each OR, and RL(N)=Luminescent count of Renilla Luciferase of each well. FIG. 34 showsthe activation patterns of the human odorant receptors in response toodiferous agent exposure.

Example 21 Cell-Surface Expression of V1RE11 in Hana3A and 293T Cells

cDNAs encoding a putative pheromone receptor (V1RE11) were transfectedinto Hana3A cells (HEK293T cells expressing REEP1, RTP1, RTP2 andG_(αolf)) or 293T cells. V1RE11 is a putative pheromone receptor in themouse and is completely different from odorant receptors in amino acidsequences. Hana3A cells supported cell-surface expression of V1RE11. Thepresent invention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, these results indicate that REEP1, RTP1, andRTP2 can support functional expression of receptors other than odorantreceptors.

Example 22 The Ability Of RTP1-A, RTP1-B, RTP1-C, RTP1-D And RTP1-E toEnhance OLFR62 Cell-Surface Expression and Activity

This example describes the generation of the RTP1 variants RTP1-A,RTP1-B, RTP1-C, RTP1-D and RTP1-E and their ability to enhance OLFR62cell-surface expression and activity. Variants of RTP1 were generated bydeleting portions of RTP1. FIG. 35 schematically shows the amino acidsegments of RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E in comparison toRTP1. pCI was a control vector. FIG. 36 shows the murine amino acidsequence for RTP1-A (SEQ ID NO: 41), FIG. 37 shows the murine amino acidsequence for RTP1-B (SEQ ID NO: 42), FIG. 38 shows the murine amino acidsequence for RTP1-C (SEQ ID NO: 43), FIG. 39 shows the murine amino acidsequence for RTP1-D (SEQ ID NO: 44), and FIG. 40 shows the murine aminoacid sequence for RTP1-E (SEQ ID NO: 45).

FIG. 41 shows cell-surface expression of OLFR62 in Hana3A and 293Tcells. cDNAs encoding RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, andcontrol pCI were transfected into Hana3A cells or 293T cells. Increasedcell-surface staining was seen in Hana3A cells and 239T cells expressingRTP1-D.

FIG. 42 schematically shows a luciferase assay used to monitor theactivity of OLFR62 activity. cAMP responsive element (CRE) andluciferase was used to monitor activation of OLFR62. Activation ofOLFR62 increases cAMP, which enhances the expression of luciferasereporter gene through the CRE.

FIG. 43 shows OLFR62 activity as indicated by luciferase expression inHana3A cells and 293T cells expressing RTP1, RTP1-A, RTP1-B, RTP1-C,RTP1-D, RTP1-E, and control pCI. Increased enhancement of OLFR62activity was seen in 293T cells and Hana3A cells expressing RTP1-D.

Example 23 The Ability of RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 toEnhance OLFR62 Cell-Surface Expression and Activity

This example describes the generation of the RTP1 variants RTP1-A1,RTP1-D1, RTP1-D2, and RTP1-D3 and their ability to enhance OLFR62cell-surface expression and activity. Variants of RTP1 were generated bydeleting portions of RTP1-A and RTP1-D. In particular, primer pairs werelaid down at specific locations corresponding to desired deletionsegments and were amplified by PCR with KOD polymerase. FIG. 44schematically shows the amino acid segments of RTP1-A1, RTP1-D1,RTP1-D2, and RTP1-D3 in comparison to RTP1-A and RTP1-D, respectively.FIG. 45 shows the murine amino acid sequence for RTP1-A1 (SEQ ID NO: 46)and the human amino acid sequence for RTP1-A1 (SEQ ID NO: 47). FIG. 46shows the murine amino acid sequence for RTP1-D1 (SEQ ID NO: 48). FIG.47 shows the murine amino acid sequence for RTP-D2 (SEQ ID NO: 49). FIG.48 shows the murine amino acid sequence for RTP-D3 (SEQ ID NO: 50).

FIG. 49 shows cell-surface expression of OLFR62 in 293T cells. cDNAsencoding RTP1, RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, and control pCIwere transfected into 293T cells. Increased cell-surface staining wasseen in 239T cells expressing RTP1-A1, RTP1-D1 and RTP1-D3.

FIG. 50 shows OLFR62, OREG, S6, and 23-1 activity as indicated byluciferase expression in 293T cells expressing RTP1, RTP1-A1, RTP1-D1,RTP1-D2, and RTP1-D3, and control pCI. Increased enhancement of OLFR62,OREG, S6, and 23-1 activity was seen in 293T cells expressing RTP1-A1.

FIG. 51 shows OLFR62, OREG, S6, and 23-1 activity as indicated byluciferase expression in Hana3A cells expressing RTP1, RTP1-A1, RTP1-D1,RTP1-D2, and RTP1-D3, and control pCI.

FIG. 52 shows cell-surface expression of OLFR62, OREG, MOR203-1, S6, and23-1 in 293T cells co-transfected with either RTP1, RTP1-A1 or controlpCI. cDNAs encoding RTP1, RTP1-A1, and control pCI were transfected intocells. Increased cell-surface staining was seen in cells expressingRTP1-A1.

Example 24 RTP1 and RTP4 Chimeras

This example describes RTP1 and RTP4 chimeras generated with chimericPCR. In particular, complex chimera primers were designed at theconnection points of the RTP1 and RTP4 sequences. For each chimera, twopairs of primers were first amplified (e.g., forward primer and complexprimer, complex primer and reverse primer). Next, the two PCR productswere used as templates in a subsequent megaprimer PCR, with the originalforward and reverse primers, to obtain a desired chimera. FIG. 53schematically shows the amino acid segments of RTP1-A1-A (Chimera 1),RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4),RTP14-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6).

FIG. 54 shows cell-surface expression of an OR in cells expressing RTP1,RTP4, Chimera 1, Chimera 2, Chimera 3, Chimera 4, Chimera 5, Chimera 6,and control pCI. cDNAs encoding RTP1, RTP4, RTP1-A1, Chimera 1, Chimera2, Chimera 3, Chimera 4, Chimera 5, Chimera 6, and control pCI weretransfected into 293T cells.

FIG. 55 shows OLFR62, OREG, S6, and 23-1 activity as indicated byluciferase expression in 293T cells expressing RTP1, RTP4, RTP1-A1,RTP1-D1, RTP1-D2, Chimera 1, Chimera 2, Chimera 3, Chimera 4, Chimera 5,Chimera 6, and control pCI.

FIG. 56 shows detection of RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-A1,RTP1-D, Chimera 4, Chimera 5, RTP1-D3, RTP1-D1, Chimera 6, and RTP4using anti-RTP1.

All publications and patents mentioned in the above specification areherein incorporated by reference. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

1. A method for identifying an odorant receptor ligand, comprising: a)providing i) a cell comprising an odorant receptor, wherein said cellfurther comprises a first heterologous nucleic acid encoding apolypeptide comprising an amino acid sequence that is at least 80%identical to SEQ ID No. 37, wherein said polypeptide is capable ofpromoting odorant receptor cell surface localization and odorantreceptor functional expression, and a second heterologous nucleic acidencoding a polypeptide comprising an amino acid sequence that is atleast 80% identical to SEQ ID No. 38, wherein said polypeptide iscapable of promoting odorant receptor cell surface localization andodorant receptor functional expression, ii) at least one test compound;b) exposing said test compound to said cell; and c) detecting theactivity of said odorant receptor.
 2. The method of claim 1, whereinsaid at least one test compound comprises more than one test compound.3. The method of claim 1, wherein said detecting comprises detecting areporting agent.
 4. The method of claim 1, wherein said odorant receptoris a human odorant receptor.
 5. The method of claim 1, wherein said testcompound is an odoriferous molecule.
 6. The method of claim 1, whereinsaid odorant receptor is a murine odorant receptor.
 7. The method ofclaim 1, wherein said odorant receptor is a synthetic odorant receptor.8. The method of claim 1, wherein said at least one test compound isexposed in the presence of a reference compound previously identified asa ligand for said odorant receptor.
 9. The method of claim 1, whereinsaid at least one test compound corresponds to a mixture of differenttest compounds.
 10. The method of claim 1, further comprising the stepof d) detecting the presence or absence of an odorant receptor ligandbased upon said activity.
 11. A method for identifying an odorantreceptor ligand, comprising: a) providing i) a cell comprising anodorant receptor, wherein said cell further comprises a firstheterologous nucleic acid encoding a polypeptide comprising an aminoacid sequence that is at least 80% identical to SEQ ID No. 47, whereinsaid polypeptide is capable of promoting odorant receptor cell surfacelocalization and odorant receptor functional expression, and a secondheterologous nucleic acid encoding a polypeptide comprising an aminoacid sequence that is at least 80% identical to SEQ ID No. 38, whereinsaid polypeptide is capable of promoting odorant receptor cell surfacelocalization and odorant receptor functional expression, ii) at leastone test compound; b) exposing said test compound to said cell; and c)detecting the activity of said odorant receptor.
 12. The method of claim11, wherein said at least one test compound comprises more than one testcompound.
 13. The method of claim 11, wherein said detecting comprisesdetecting a reporting agent.
 14. The method of claim 11, wherein saidodorant receptor is a human odorant receptor.
 15. The method of claim11, wherein said test compound is an odoriferous molecule.
 16. Themethod of claim 11, wherein said odorant receptor is a murine odorantreceptor.
 17. The method of claim 11, wherein said odorant receptor is asynthetic odorant receptor.
 18. The method of claim 11, wherein said atleast one test compound is exposed in the presence of a referencecompound previously identified as a ligand for said odorant receptor.19. The method of claim 11, wherein said at least one test compoundcorresponds to a mixture of different test compounds.
 20. The method ofclaim 11, further comprising the step of d) detecting the presence orabsence of an odorant receptor ligand based upon said activity.